Flt3-L mutants and methods of use

ABSTRACT

A screening method for identifying mutant polypeptides having at least one amino acid difference from a wild type protein involved in a receptor-ligand interaction is disclosed. Also disclosed are mutant polypeptides of the hematopoietic growth factor flt3-Ligand (flt3-L) identified using this method, nucleic acids encoding these flt3-L mutant polypeptides, and methods of treatment involving in vitro and in vivo use of the mutant polypeptides and nucleic acids.

FIELD OF THE INVENTION

[0001] The invention relates to mutant polypeptides in which proteinresidues involved in receptor-ligand interactions have been altered, andnucleic acids encoding these polypeptides.

BACKGROUND OF THE INVENTION

[0002] Flt3 Ligand (flt3-L) is a protein that binds to a cell surfacetyrosine kinase, flt3 Receptor (flt3). The human flt3 gene has beencloned, and encodes a protein belonging to a family of structurallyrelated tyrosine kinase receptors that contain five extracellularimmunoglobulin (Ig)-like domains and an intracellular tyrosine kinasedomain (Small et al., Proc. Natl. Acad. Sci. 91:459-463 (1994)). Whileflt3 is expressed in a limited number of tissues, including human bonemarrow, thymus, spleen, liver, and lymph nodes, flt3-L is widelyexpressed in human tissue (Brasel, et al., Leukemia 9:1212-1218 (1995);Lyman, et al., Blood 83:2795-2801 (1994)).

[0003] Structural studies have demonstrated that human flt3-L is amember of the four helix bundle protein family of cytokines. The humanflt3-L gene encodes a 235 amino acid type I transmembrane proteinconsisting of four domains: an amino-terminal 26 residue signal peptide;a 156 residue extracellular domain; a 23 amino acid transmembranedomain; and a 30 residue cytoplasmic domain (Hannum et al., Nature368:643-648 (1994); Lyman et al., Cell 75:1157-1167 (1993); Lyman etal., Blood 83:2795-2801 (1994)). The amino terminal 26 residue signalpeptide is cleaved from the full length polypeptide to yield the matureprotein. Soluble flt3-L, which is thought to be released intocirculation from the cell membrane by protease cleavage (Lyman et al.,Oncogene 10:147-149 (1995)), is a noncovalently linked dimer containingsix cysteine residues that apparently form intramolecular disulfides.Flt3-L is similar in size and structure to other four-helixhematopoietic growth factors such as Stem Cell Factor (SCF; also knownas mast cell growth factor, Steel Factor (SF), and kit ligand) andmacrophage colony stimulating factor (M-CSF), also known as colonystimulating factor I (CSF I), which also bind to and activate tyrosinekinase receptors. Despite their structural similarities, however, thesethree growth factors have very little conserved primary sequence.

[0004] The nature of flt3-L binding to flt3 has not been fullycharacterized previously. Site-directed mutagenesis has been used tostudy the structure and function of proteins, when the region of theprotein to be mutagenized is already defined. However, in the case ofcertain proteins, such as flt3-L, the region of interest in the protein,e.g., the region that binds to flt3, is not well defined. The crossreactivity of murine and human flt3-L for flt3 (Lyman et al., Blood83:2795-2801 (1994)) precludes the potential of identifying residues ofinterest by swapping interspecies segments of polypeptide between theseligands. Comprehensive mutational studies of some of the other membersof the four helix bundle protein family may not be applicable to flt3-L,because a number of these species are monomeric and bind class Ihematopoietic receptors, whereas native flt3-L forms a dimer, and bindsto and activates a class III tyrosine kinase receptor.

[0005] Studies of flt3-L function indicate that its binding to flt3initiates a signaling event that regulates the proliferation anddifferentiation of multiple lineages of cells of the hematopoieticsystem (Hannum et al., Nature 368:643-648 (1994); Lyman et al., Cell75:157-1167 (1993); for review see Lyman, Int. J. Hemat. 62:63-73(1995)). In combination with other growth factors, flt3-L has potentsynergistic proliferative effects on hematopoietic precursor or stemcells (Hannum et al., Nature 368:643-648 (1994); Jacobsen et al., J.Exp. Med. 181:1357-1363 (1995)). Flt3-L can also induce theproliferation of other cell types, including T cells, early B cells anderythroid cells (U.S. Pat. No. 5,554,512).

[0006] SCF and M-CSF also activate hematopoietic cells. M-CSF primarilyactivates cells of the monocyte-macrophage lineage, while SCF acts on anumber of cell lineages in both the lymphoid and myeloid pathway, aswell as on primitive hematopoietic cells. Unlike flt3-L, SCF alsostimulates proliferation and activation of mast cells, which producehistamine and can cause anaphylactic reactions in vivo. Intravenousadministration of SCF in mice results in a respiratory distress syndromecharacterized by breathing difficulties believed to result fromdegranulation of mast cells in the lungs. In contrast, flt3-L does notinduce respiratory distress in mice following the injection of a largeintravenous dose. See Lyman, Int. J. Hematol. 62:63-73 (1995).

[0007] In addition to its ability to induce cellular proliferation,flt3-L can induce the differentiation of hematopoietic progenitor cells,i.e., CD34⁺ bone marrow progenitors and stem cells, into other celltypes, including myeloid precursor cells, monocytic cells, macrophages,B lymphocytes, natural killer (NK) cells and dendritic cells. Dendriticcells can be used to present antigens, including tumor and viralantigens, to naive T cells, and can also be used as vaccine adjuvants,i.e., facilitators of immune responses to vaccines. See, e.g., WO97/12633. Previously, the use of dendritic cells as immunostimulatoryagents or adjuvants was limited by the low frequency of dendritic cellsin peripheral blood, the limited accessibility to lymphoid organs, andthe terminal state of differentiation of dendritic cells. Sincedendritic cells are antigen-presenting cells, an increase in thedendritic cell population in vivo could augment presentation of antigensincluding tumor, bacterial and viral antigens to T cells.

[0008] Flt3-L's ability to regulate the growth and differentiation ofhematopoietic progenitor cells indicates that it would be clinicallyuseful in treating hematopoietic disorders, including aplastic anemiaand myelodysplasia. Flt3-L can also be used to enhance populations ofcertain cell types in patients undergoing allogeneic, syngeneic orautologous bone marrow transplantation procedures having cytoreductiveeffects. See U.S. Pat. No. 5,554,512. For example, the use of ionizingradiation or chemical toxins to treat neoplasia results in cytotoxiceffects on normal as well as cancerous cells. These therapies can causemyelosuppression, i.e., damage to bone marrow cells that are theprecursors of cells including lymphocytes, erythrocytes and platelets.Myelosuppression results in cytopenia, i.e., blood cell deficits, thatincrease the risk of infection and bleeding disorders. One approach tothe treatment of cytopenias is the removal of hematopoietic cells from apatient prior to cytoreductive therapies, and infusion of the cells backinto the patient after therapy, to restore hematopoietic cell function.Since flt3-L induces proliferation of hematopoietic cells, it can beused in vitro to expand the population removed from the patient, and theexpanded cell population can then be administered to the patient.Because flt3-L will also induce hematopoietic progenitor cells todifferentiate into NK cells and dendritic cells, it can be administeredto patients in need of expanding their NK cell or dendritic cellsubpopulations, and used in vitro, to induce differentiation of isolatedhematopoietic cells into NK and dendritic cells, which can then beadministered to a patient.

[0009] Flt3-L's ability to induce the proliferation or differentiationof certain cell types indicates that it has therapeutic significance forother conditions, including Acquired Immune Deficiency Syndrome (AIDS)and human immunodeficiency virus (HIV) infection, and cancers, includingbreast cancer, lymphoma, small cell lung cancer, multiple myeloma,neuroblastoma, leukemias, testicular cancer and ovarian cancer.

[0010] Since flt3-L is known to induce proliferation and differentiationof certain cell types, it would be advantageous to develop methods ofincreasing or decreasing flt3-L function for therapeutic applications.One method of accomplishing this goal would be to characterize therelationship of flt3-L with its receptor, flt3, to determine whichregions of flt3-L are implicated in ligand binding and biologicalactivity, to develop flt3-L mutants with increased or decreasedactivity. The characteristics of flt3-L-flt3 binding and theascertainment of the residues necessary for the induction of thebiological effects attributed to the binding of flt3-L to flt3 have notbeen defined previously. The derivation of mutant forms of flt3-L whicheither augment or decrease the biological activity of flt3-L would beuseful in designing therapeutic strategies for modulation of flt3-Lactivity to treat a variety of pathological conditions.

SUMMARY OF THE INVENTION

[0011] The invention includes a screening method for identifying mutantpolypeptides in which at least one amino acid residue of a proteininvolved in a receptor-ligand interaction has been altered; isolatedmutant polypeptides identified using this method; nucleic acids encodingthese mutant polypeptides; and methods of treatment involvingadministration of these mutant polypeptides and nucleic acids. Themethod of screening described herein to identify protein residuesinvolved in receptor-ligand interaction has allowed the definition ofregions of interaction between flt3 Ligand (flt3-L) and its cognatereceptor (flt3), and the identification and isolation of flt3-L mutantswith altered biological activity. Using this information, polypeptideshaving multiple amino acid substitutions relative to the wild type humanflt3-L have been constructed. The flt3-L mutant polypeptides and nucleicacids described herein are useful for in vitro applications, as well astherapeutically in vivo.

[0012] The invention includes a substantially pure flt3-L mutant proteinor polypeptide. Flt3-L mutant polypeptides are preferably derived from amammal, such as a mouse or a human.

[0013] The terms “protein” and “polypeptide” are used interchangeablyherein, and refer to any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). Proteins or polypeptides of the invention arepreferably at least 5 amino acids in length.

[0014] The full-length, wild type human flt3-L polypeptide sequence isdisclosed in U.S. Pat. No. 5,554,512, and is set forth herein as SEQ IDNO:1. Amino acids one to twenty-six are cleaved from the full lengthprotein to give the mature flt3-L protein, starting at the threonineresidue at position 27 of the full length wild type protein. Thesequence of the mature human wild type flt3-L polypeptide is set forthas SEQ ID NO:18. A “mutant flt3-L polypeptide” is a polypeptide having asequence that has at least one difference in amino acid sequencerelative to a wild type flt3-L polypeptide. Such a difference in aminoacid sequence may arise, e.g., by substitution of one amino acid foranother, or by deletion or addition of amino acids. These differences inamino acid sequence occur, e.g., within the regions encompassed bypositions 8-15, 81-87 and 116-124 of the mature wild type human flt3-Lprotein (SEQ ID NO:18), and include proteins that differ from the maturewild type human flt3-L protein (SEQ ID NO:18) by amino acidsubstitutions at position 8, 84, 118 or 122.

[0015] Other mutant flt3-L polypeptides differ from wild type humanflt3-L by amino acid substitutions that affect the dimerizationinterface of the protein. Such substitutions are at, e.g., positions 26,27 or 64 of the mature wild type flt3-L polypeptide (SEQ ID NO:18). The“dimerization interface” of a protein includes those regions of theprotein that physically contact each other when the protein is in itswild type dimeric form. Mutant flt3-L polypeptides also include flt3-Lmutant ligands in which amino acid substitutions occur in regionsoutside of the mature protein. Such substitutions include, e.g., asubstitution at the -3 position of the mature wild type human flt3-Lpolypeptide, i.e., at position 24 of the full length wild type humanflt3-L (SEQ ID NO:1).

[0016] Mutant flt3-L polypeptides also include those having an alteredcharge distribution from a wild type flt3-L polypeptide. Such an alteredcharge distribution can result in altered flt3-L biological activity.For example, substitution of amino acid residues in the region of aminoacid positions 118-124 of the mature flt3-L polypeptide (SEQ ID NO:18),e.g., at positions 118 or 122 of the mature flt3-L protein, with basicresidues can produce a flt3-L mutant polypeptide with increased flt3-Lbiological activity. Basic residues can also be added to a wild typeflt3-L polypeptide to produce a mutant flt3-L polypeptide with alteredbiological activity. Substitution of a basic residue in the region ofposition 81-87 of mature wild type flt3-L, e.g., substitution of thelysine residue at position 84 of the mature wild type protein, or anincrease in net negative charge relative to wild type flt3-L, can alsoresult in a flt3-L polypeptide with increased biological activity.

[0017] Mutant flt3-L polypeptides can be identified by, e.g., using thescreening assay described herein. Once identified, the mutantpolypeptides can be generated by conventional methods, e.g., techniquessuch as site-directed mutagenesis of appropriate nucleic acid sequencesand expression of the mutant proteins in standard expression systems.

[0018] Mutant flt3-L polypeptides include “multiple mutant flt3-Lpolypeptides,” i.e., flt3-L mutant polypeptides having more than onedifference in amino acid sequence relative to a wild type flt3-Lpolypeptide. For example, a multiple mutant flt3-L polypeptide has twoor more amino acid substitutions relative to the wild type human flt3-Lpolypeptide. Multiple mutant flt3-L polypeptides can be generated by,e.g., subcloning nucleic acid fragments containing appropriatemutations, or by site-directed mutagenesis of appropriate nucleic acidsequences, and expression of the mutant protein in a standard expressionsystem. Multiple mutant flt3-L polypeptides include those havingmutations affecting the dimerization interface as well as mutationsaffecting receptor binding affinity or induction of cellularproliferation or differentiation.

[0019] A “substantially identical” polypeptide sequence differs from agiven sequence only by conservative amino acid substitutions or by oneor more nonconservative substitutions, deletions, or insertions locatedat positions which do not destroy the biological activity of thepolypeptide.

[0020] A “substantially pure” preparation is at least 60% by weight ofthe compound of interest, e.g., a flt3-L mutant polypeptide. Preferablythe preparation is at least 75%, more preferably at least 90%, and mostpreferably at least 95% of the compound of interest. Purity of thecompound can be assessed by appropriate methods that are well known inthe art, e.g., column chromatography, polyacrylamide gelelectrophoresis, or High Performance Liquid Chromatography (HPLC).

[0021] Polypeptides of the invention include, but are not limited to,recombinant polypeptides, natural polypeptides, and syntheticpolypeptides, as well as preproteins or proproteins.

[0022] Polypeptides of the invention include those that have beenmodified to facilitate their uptake by cells, e.g., by packing intoliposomes.

[0023] A polypeptide of the invention also includes those that have beenphysically linked to another polypeptide, e.g., a marker polypeptide.For example, the polypeptide is fused to a hexa-histidine tag tofacilitate purification of bacterially expressed proteins, or ahemagglutinin tag to facilitate purification of proteins expressed ineukaryotic cells.

[0024] Soluble flt3-L mutant polypeptides are also included in theinvention. These soluble polypeptides include those in which all or apart of the transmembrane portion of the polypeptide has been removed.The remainder of the protein may form a fusion protein with anothersoluble polypeptide or another cytokine such as erythropoietin (EPO),thrombopoietin (TPO), GM-CSF, G-CSF, members of the interleukin (“IL”)family, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, or IL-15, or a fragment thereof.Alternatively, the other soluble polypeptide is an immunoglobulin Fcdomain. Such fusion proteins are readily purified using a protein Acolumn.

[0025] A “biologically active” polypeptide of the invention possessesany biological activity characteristic of flt3-L. Biological activitiescharacteristic of flt3-L, include, but are not limited to, being capableof binding flt3, or transducing a stimulatory signal to a cell throughmembrane-bound flt3, resulting in effects such as cellular proliferationor differentiation.

[0026] A mutant flt3-L polypeptide exhibits increased or decreasedflt3-L biological activity relative to a wild type flt3-L polypeptide.

[0027] “Increased” flt3-L biological activity is characterized by anincrease of at least about 40% of a biological activity of wild typeflt3-L, such as receptor binding affinity or induction of cellularproliferation or differentiation. Such an increase in biologicalactivity can be measured using conventional techniques.

[0028] “Decreased” flt3-L biological activity is characterized by adecrease of at least about 40% of a biological activity of wild typeflt3-L, such as receptor binding affinity or induction of cellularproliferation or differentiation. Such a decrease in biological activitycan be measured using conventional techniques.

[0029] Another embodiment of the invention is a purified polynucleicacid that comprises a sequence encoding a flt3-L mutant polypeptide, asoluble flt3-L mutant polypeptide, or a fragment of such a polypeptide.Preferably, the nucleic acids are derived from a mammal. The sequence ofa wild type human flt3-L cDNA is disclosed in U.S. Pat. No. 5,554,512,and is set forth herein as SEQ ID NO:2. A “flt3-L mutant nucleic acid”is a polynucleic acid encoding a flt3-L mutant polypeptide or protein,as described above. Such mutant flt3-L nucleic acids have undergone oneor more insertions, deletions, substitutions or other mutations, orcombinations thereof, relative to a wild type flt3-L polynucleic acid.Flt3-L mutant nucleic acids include “flt3-L multiple mutant nucleicacids” i.e., a polynucleic acid encoding flt3-L multiple mutantpolypeptides, as described above.

[0030] The term “nucleic acid” encompasses both RNA and DNA, includingcDNA, genomic DNA and synthetic (e.g., chemically synthesized) DNA. Thenucleic acid is double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid is the sense strand or the antisensestrand. Polynucleic acids of the invention include a recombinant nucleicacid incorporated into a vector, such as an autonomously replicatingplasmid or virus; a cDNA or genomic DNA fragment produced by polymerasechain reaction (PCR) or restriction endonuclease treatment; orrecombinant DNA that is part of a hybrid gene encoding additionalpolypeptide sequences.

[0031] An “isolated” molecule, such as a polypeptide or a nucleic acid,is free from association with at least some proteins that the respectivenative molecules are associated with in their natural environment. Forexample, an isolated polypeptide may be a purification product of arecombinant host cell culture, or a purified extract.

[0032] The invention also includes transfected or transformed cellsharboring a nucleic acid described herein. Vectors and plasmids thatinclude a nucleic acid properly positioned for expression are alsowithin the invention.

[0033] A “transfected cell” or “transformed cell” is a cell into which(or into an ancestor of which) a nucleic acid of the invention has beenintroduced.

[0034] “Positioned for expression” means that the selected nucleic acidmolecule is positioned adjacent to one or more sequence elements whichdirect transcription or translation of the sequence of the selectednucleic acid (i.e., the selected nucleic acid is operably associatedwith the sequence elements).

[0035] The flt3-L mutant proteins, including soluble mutant and multiplemutant flt3-L proteins, and nucleic acids of the invention are used toprepare pharmaceutical compositions to be used in methods of allogeneic,syngeneic or autologous transplantation. Pharmaceutical compositionscomprise flt3-L mutant proteins or nucleic acids alone, or incombination with other growth factors or nucleic acids encoding suchgrowth factors. Growth factors used with flt3-L include, but are notlimited to, interleukins, colony stimulating factors and proteinkinases.

[0036] The invention also includes a method of inducing proliferation ofhematopoietic progenitor or stem cells using flt3-L mutant or multiplemutant proteins. The method includes the steps of isolating a populationof cells to be expanded and exposing them in vitro to a mutant flt3-Lpolypeptide. The expanded cell population is then introduced into apatient. The population of cells is, for example, hematopoietic cells.

[0037] A method of expanding a population of cells in vivo is alsoincluded in the invention. According to the method, a pharmaceuticalcomposition of a mutant or multiple mutant flt3-L polypeptide or nucleicacid sufficient to induce proliferation of a target cell population isadministered to a patient.

[0038] The invention also includes a method of modulating an immuneresponse in a subject, by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition that includes a flt3-Lmutant polypeptide or nucleic acid.

[0039] The invention further includes a method of treating an immunedisorder in a patient by administration of a therapeutically effectiveamount of a pharmaceutical composition comprising a flt3-L mutantpolypeptide or nucleic acid. Such immune disorders include, but are notlimited to, allergy, immunosuppression, and autoimmunity.

[0040] A method of treating a pathological condition by administrationof a pharmaceutical composition of a flt3-L mutant polypeptide ornucleic acid is also included in the invention. Such pathologicalconditions include, but are not limited to, myelodysplasia, aplasticanemia, HIV infection and cancer, including breast cancer, lymphoma,small cell lung cancer, multiple myeloma, neuroblastoma, acute leukemia,testicular cancer and ovarian cancer.

[0041] The invention also includes a method of inducing cellulardifferentiation by exposure to flt3-L mutant polypeptides and nucleicacids. The method includes the steps of isolating a target population ofcells and administering an amount of flt3-L mutant polypeptidesufficient to induce the production of differentiated cells. The targetpopulation of cells is, e.g., hematopoietic cells, and thedifferentiated cells are, e.g., Natural Killer (NK), dendritic cells orfacilitating cells. Populations of differentiated cells are thenintroduced into a subject in need of such cells. Alternatively, themethod involves the in vivo administration of flt3-L mutant or multiplemutant polypeptides to a patient.

[0042] Induction of “cellular differentiation” is the induction of cellsto differentiate along certain lineages. For example, hematopoieticcells can differentiate into cell types including NK cells, dendriticcells, and facilitating cells.

[0043] The invention also includes a method of augmenting an immuneresponse in a patient, by administering an amount of a flt3-L mutantpolypeptide sufficient to generate an increase in the number of thepatient's dendritic cells. The patient can have an infectious disease,such as HIV, or a cancerous neoplastic disease.

[0044] A method of enhancing a mammal's immune response to a vaccineantigen is also included in the invention. The method includes the stepsof administering an immunogenic amount of the vaccine antigen and animmunogenicity-augmenting amount of a flt3-L mutant polypeptide, inconcurrent or sequential combination with the vaccine antigen.

[0045] An “adjuvant” is a substance that enhances, augments orpotentiates a host's immune response to a vaccine antigen.“Immunogenicity” is the ability of an immunogen or antigen to provoke animmune response in a subject.

[0046] A “therapeutically effective amount” of a substance is an amountcapable of producing a medically desirable effect in a treated subject.

[0047] The invention also includes a screening method for identifyingresidues involved in receptor binding in a receptor-ligand system. Themethod includes the steps of:

[0048] subjecting a nucleic acid population encoding the ligand torandom mutagenesis, to form a mutagenized ligand population;

[0049] transforming cells with the mutagenized ligand population, toform transformed colonies;

[0050] transferring the transformed colonies to a first membrane;

[0051] overlaying the first membrane with a second membrane, the secondmembrane being coated with capture means for capturing the ligand andmutants thereof;

[0052] reacting the second membrane with a receptor for the ligand; and

[0053] subsequently reacting the second membrane with detection meansfor detecting receptor binding to the ligand or mutants thereof.

[0054] The transformed cells can be, e.g., yeast or bacterial cells.

[0055] The invention also includes a method of screening to identifymutant polypeptides with altered expression characteristics. This methodincludes the steps of:

[0056] subjecting a nucleic acid population encoding the ligand tomutagenesis, to form a mutagenized ligand population;

[0057] transforming cells with the mutagenized ligand population, toform transformed colonies;

[0058] transferring the transformed colonies to a first membrane;

[0059] overlaying the first membrane with a second membrane, the secondmembrane being coated with capture means for capturing the ligand andmutants thereof;

[0060] reacting the second membrane with a receptor for the ligand; and

[0061] subsequently reacting the second membrane with means fordetecting receptor binding to the ligand or mutants thereof.

[0062] The transformed cells can be, e.g., yeast or bacterial cells.

[0063] A “receptor-ligand system” is a system in which a ligand binds toa receptor that specifically recognizes it. As used herein, “capturemeans” include any means that can be used for capturing a specificligand. Such capture means include, for example, an antibody thatspecifically recognizes and binds a particular ligand. “Detection means”include any means that can be used for the detection of receptor-ligandbinding. Such detection means include, for example, an antibody thatspecifically recognizes and binds a particular receptor bound to animmobilized ligand, and fluorescent or enzymatic means to detect thebound receptor.

[0064] The invention also includes mutant M-CSF and SCF polypeptides,and nucleic acids encoding them. Such mutant polypeptides have sequenceswith at least one difference in amino acid sequence from their wild typecounterparts, in regions that correspond to key positions involved inreceptor-ligand binding. These regions include the regions correspondingto the regions defined by amino acid positions 8-15, 81-87 and 116-124of the mature human wild type flt3-L polypeptide when the amino acidsequences of flt3-L, SCF and M-CSF are aligned. For example, suchmutants include those in which there is an amino acid difference at theposition corresponding to position 8 in mature wild type flt3-L, e.g.,position 9 of M-CSF.

[0065] The mutant SCF and M-CSF polypeptides include those with aminoacid differences that cause the mutant polypeptides to lose affinity fortheir receptors, and instead bind a different receptor. For example, theinvention includes a mutant SCF polypeptide which binds to and activatesflt3-expressing cells, but which does not bind c-kit.

[0066] The invention further includes small molecules in which the keyresidues involved in flt3 binding, as defined herein, or functionalgroups corresponding to the side chains of these residues, have beeninserted. Such residues include, e.g., those within the regions definedby amino acid positions 8-15, 81-87 and 116-124, and amino acidpositions 26, 27 and 64, of the mature human flt3-L wild typepolypeptide. Functional groups which correspond to the side chains ofamino acids are well known to those in the art and include, e.g.,substitution of an amine functional group for a lysine residue. A “smallmolecule” is a molecule that acts as a scaffold by maintaining a threedimensional structure allowing flt3-L binding to and activation offlt3-expressing cells.

[0067] Additional objects and advantages of the invention will be setforth in part in the description that follows, and in part will beapparent from the description, or may be realized during the practice ofthe invention. The objects and advantages of the invention will berealized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. Both the foregoinggeneral description and the following detailed description are exemplaryand explanatory only, and are not restrictive of the claimed invention.All publications, patent applications and other references mentionedherein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068]FIG. 1 is an amino acid sequence alignment of the human and murineflt3 Ligand polypeptide (flt3-L) with the human M-CSF polypeptide.

[0069]FIG. 2 is a representation of the secondary (top) and primary(bottom) structures of flt3-L, showing the specific activity of isolatedflt3-L mutant polypeptides relative to the wild type flt3-L polypeptide.

[0070]FIG. 3 shows the results of flt3 Receptor (flt3) binding andBAF/hflt3 cell proliferation assays of flt3-L mutant polypeptidesrelative to wild type flt3-L polypeptide.

[0071]FIGS. 4A and 4B are three dimensional representations of theflt3-L polypeptide.

[0072]FIGS. 5A and 5B are size exclusion chromatographs of wild type (A)and mutant (B) flt3-L proteins at different concentrations.

DETAILED DESCRIPTION

[0073] Flt3-L mutant polypeptides, including multiple mutantpolypeptides, having altered biological activity have been identifiedand isolated. In addition, novel mutants have been constructed thatexhibit increased or decreased flt3-L biological activity. The flt3-Lmutants described herein can be used in vitro, or in vivo, inpharmaceutical compositions, to modulate the effects of flt3-L-flt3binding to treat a variety of pathological conditions. In addition, themethod used to identify these mutants is applicable to anyligand-receptor system for which appropriate biological assays can bederived.

[0074] To identify flt3-L mutants, yeast colonies were transformed withDNA encoding wild type flt3-L that had been randomly mutagenized. Thetransformed colonies were lifted onto a first membrane, which wasoverlaid by a second or “capture” membrane. The second membrane wascoated with an antibody to the ligand, to form a membrane “sandwich.”Ligand protein secreted by the yeast passed through the first membrane,and was captured by the antibody immobilized on the second membrane. Thesecond membrane was then probed with the receptor for the ligand, andmeans were used to detect receptor-ligand binding.

[0075] This method was used to screen a randomly mutagenized ligand,flt3-L, to identify the residues involved in binding to its receptor,flt3. Sixty thousand colonies were screened, and DNA from a subset of 59clones was sequenced. Thirty-one single amino acid substitutions at 24positions throughout the primary sequence of flt3-L either enhanced orreduced activity in receptor binding and cell proliferation assays.Representative flt3-L mutant proteins were purified and analyzed forreceptor binding, specific activity, Stokes radius and helical content.

[0076] A structural model of wild type flt3-L was generated by aligningits sequence with that of M-CSF, a member of the four-helix bundleprotein family (FIG. 1), and using the x-ray structure of M-CSF as amodel. The model, which predicts that flt3-L is a dimer, has allowed thegrouping of mutations identified using the screening method. Two typesof mutants were identified, those that are directly involved inflt3-flt3-L binding, and those that are not directly involved in thebinding interaction. While amino acid substitutions that alter flt3-Lactivity were found at sites that are scattered throughout the primaryflt3-L sequence, most of these sites were tightly grouped when displayedon the structural model of flt3-L. The residues of flt3-L implicated inreceptor binding map to a patch of the molecule defined by a regionencompassing the amino terminus of the molecule and the amino-terminalportion of helix A, the C terminus of helix C, the C terminus of helix Dand the disulfide-constrained loop that is C-terminal to helix D. Thus,the method described herein has allowed identification of the keyresidues involved in flt3 binding.

[0077] Other mutants were identified that were not directly involved inreceptor binding. These mutants had an altered structure or chargedistribution from that of wild type flt3-L. For example, the structuralmodel of flt3-L predicts that the protein is dimeric form, and mutantswere identified which interfered with the predicted dimerizationinterface, resulting in a monomeric form of flt3-L. One of these mutantswas expressed at a high level, but had low affinity for flt3. Flt3-Lmutant polypeptides having both mutations that affect dimerization andmutations that increase the affinity of flt3-L for flt3 can beconstructed using methods described herein. Such mutants would result inflt3-L mutant polypeptides which bind to but do not crosslink cellsurface flt3 molecules. Since crosslinking is crucial to cellularactivation, these combined mutants can act as antagonists. Suchantagonists can be used, for example, for the treatment of acutemyelogenous leukemias (AMLs), which express flt3.

[0078] Mutants were also identified in which an additional positivecharge due to an amino acid substitution resulted in a flt3-L mutantpolypeptide with an increased binding affinity for flt3. Mutant flt3-Lpolypeptides with increased binding affinity and biological activity cantherefore be designed by substitution of wild type flt3-L amino acidresidues with basic residues, or addition of basic residues to wildtype-flt3-L. Other mutants were identified in which substitution of abasic amino acid with another amino acid resulted in an increase inflt3-L biological activity. Therefore, mutant flt3-L polypeptides inwhich a basic amino acid has been substituted with another amino acid,and which have increased biological activity, can be designed using themethods described herein.

[0079] As described herein, mutations reducing flt3-L biologicalactivity occur throughout the primary sequence of the molecule, anddifferent amino acid changes at the same position can either decrease orincrease flt3-L biological activity, such as receptor binding oractivity in cell proliferation assays. Seventeen independent isolates ofthe same flt3-L⁺⁺ mutation were obtained. These findings support thereliability of the screening method. The method can also be used toidentify residues involved in binding in other receptor-ligand systems,and to generate mutants in those systems.

[0080] The screening method has allowed the identification of flt3-Lmutant polypeptides that increase or decrease flt3-L biologicalactivity, such as flt3 binding or induction of proliferation ofhematopoietic cells, T cells or erythrocytes. These flt3-L mutantpolypeptides can be used in therapeutic methods designed to modulate invivo flt3-L activity. Flt3-L mutants can also be used to expand ordifferentiate cell populations in vitro or in vivo. Cells expanded invitro may be transplanted into a patient in need of such cells, e.g.,patients who have undergone therapies that have cytoreductive effects.Bone-marrow progenitor cells may be induced by addition of mutant flt3-Lto differentiate into dendritic cells, e.g., which may be used asvaccine adjuvants. Flt3-L mutants may be used to treat pathologicalconditions including myelodysplasia, aplastic anemia, HIV infection,immunosuppression, autoimmune disorders, allergy and malignancies,including leukemias.

[0081] In addition, since the method described herein has allowedidentification of the key residues involved in flt3-flt3-L interaction,corresponding residues can be altered in structurally related proteins,which can be used therapeutically. For example, flt3-L shares a highdegree of structural similarity to the growth factors SCF and M-CSF.Regions of these proteins corresponding to the key residues in flt3binding can be mutated to alter receptor specificity, so that, forexample, these proteins activate the same cell types as flt3-L. The keyresidues for flt3 binding as identified herein, or functional groupscorresponding to the side chains of these residues, can also beengrafted onto small molecules. An example of a functional group thatcan be used is an amine functional group, which can be used to replace alysine residue. These small molecules maintain a three-dimensionalstructure that allows binding to and activation of flt3-expressingcells.

[0082] Flt3-L Mutant Polypeptides and Nucleic Acids

[0083] Flt3-L mutant polypeptides exhibit increased or decreasedbiological activity relative to wild type flt3-L polypeptide. Thesemutant polypeptides, which include multiple mutant polypeptides, have atleast 40% of a biological activity of wild type flt3-L, such as receptorbinding or induction of cellular proliferation or differentiation.Alternatively, the mutant polypeptides exhibit a decrease of 40% or moreof a biological activity of wild type flt3-L polypeptide. Suchcomparisons are generally based on equal concentrations of the moleculesbeing compared. The comparison can also be based on the amount ofprotein or polypeptide required to reach 50% of the maximal stimulationobtainable.

[0084] Another aspect of the invention is soluble flt3-L mutantpolypeptides. These polypeptides lack the transmembrane region thatwould cause retention of the polypeptide on the cell membrane. Solubleflt3-L mutant polypeptides include those that comprise a native orheterologous signal peptide when initially synthesized to promotesecretion. The signal peptide is cleaved upon secretion of the flt3-Lmutant polypeptide from the cell. The soluble flt3-L mutant polypeptidesretain the ability to bind flt3. Soluble flt3-L mutant polypeptides canalso include the transmembrane region or part of the cytoplasmic domainor other sequences, provided that the soluble flt3-L protein can besecreted.

[0085] Soluble flt3-L mutant polypeptides are identified anddistinguished from their non-soluble membrane-bound counterparts byseparating intact cells which express the desired protein from theculture medium, e.g., by centrifugation, and assaying the culturesupernatant for the presence of the desired protein.

[0086] Soluble forms of flt3-L mutant polypeptides can be easilypurified, since the soluble proteins are secreted from the cells.Further, soluble proteins are suitable for intravenous administration.

[0087] The soluble flt3-L mutant polypeptides of the invention includethose comprising a substantial portion of the extracellular domain of anative, full length flt3-L protein. For example, a soluble flt3-L mutantpolypeptide may comprise amino acids 28 through 235 of SEQ ID NO:1. Inaddition, truncated soluble flt3-L mutant proteins comprising less thanthe entire extracellular domain of native flt3-L are included in theinvention. Such truncated soluble proteins are represented by thesequence of amino acids 28-160, 28-182 or 28-185 of the full lengthflt3-L human polypeptide, i.e., SEQ ID NO:1. Soluble flt3-L mutantpolypeptides include those comprising a heterologous signal peptide thatfunctions within specific host cells to allow protein expression, or thenative flt3-L signal peptide.

[0088] Also within the invention are fusion proteins in which a portionof a mutant flt3-L polypeptide is fused to an unrelated protein orpolypeptide (i.e., a fusion partner). The fusion partners includemoieties selected to facilitate purification, detection, orsolubilization, or to provide some other function, such as anothercytokine, interleukin or tyrosine kinase. Fusion proteins include thoseproduced by expressing a hybrid gene in which a nucleotide sequenceencoding all or a portion of flt3-L is joined in-frame to a nucleotidesequence encoding the fusion partner. Fusion partners include, but arenot limited to, the constant region (Fc) of an immunoglobulin, such asIgG; colony stimulating factors, such as GM-CSF and G-CSF; theinterleukins, including IL-1, -2, -3,-4,-5, -6, -7, -8, -9, -10, -11,-12, -13, -14,-15, -16, -17 or -18; EPO; and TPO. Fusion of the Fcportion of IgG to a flt3-L mutant polypeptide increases its stabilityand half life. Fusion proteins include those comprising the yeast afactor signal peptide, a FLAG® peptide such as those described in U.S.Pat. No. 5,011,912, and a portion of a soluble flt3-L mutant polypeptidecorresponding to the region encompassed by amino acids 28 to 235 of SEQID NO:1. Recombinant fusion proteins are expressed in and secreted from,e.g., yeast cells. The FLAG® peptide facilitates purification of theprotein. Bovine mucosal enterokinase is used to cleave the FLAG® peptidefrom the soluble flt3-L mutant polypeptide.

[0089] Flt3-L mutant polypeptide fusions comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204(1988).

[0090] The invention further includes flt3-L mutant polypeptides with orwithout associated native-pattern glycosylation. Mutant flt3-Lpolypeptides expressed in yeast or mammalian cells (e.g., COS-7 cells)are similar to or significantly different from a native flt3-Lpolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of flt3-L polypeptidesin bacterial cells, such as E. coli, results in non-glycosylatedmolecules.

[0091] Flt3-L mutant polypeptides can be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of flt3-L mutant polypeptides are prepared, e.g., by linkingthe chemical moieties to functional groups on flt3-L amino acid sidechains or at the amino terminus or carboxy terminus of a flt3-L mutantpolypeptide or the extracellular domain thereof. Other derivativesinclude covalent or aggregative conjugates of a flt3-L mutantpolypeptide or its fragments with other proteins or polypeptides, suchas by synthesis in recombinant culture as amino terminal or carboxyterminal fusions proteins. For example, the conjugate comprises a signalor leader polypeptide sequence (e.g., the α-factor leader ofSaccharomyces) at the amino terminus of a flt3-L mutant polypeptide. Thesignal or leader peptide co-translationally or post-translationallydirects transfer of the conjugate from its site of synthesis to a siteinside or outside of the cell membrane or cell wall.

[0092] Isolated nucleic acids encoding soluble flt3-L mutant proteinsare also included in the invention. Nucleic acids capable of expressingsoluble mutant flt3-L polypeptides, including truncated polypeptides,are prepared by any of a number of conventional techniques. Techniquesfor preparation of recombinant nucleic acids and expression ofpolypeptides therefrom are described in detail in Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, New York, 1994. For example,desired nucleic acid sequences are chemically synthesized using knowntechniques or produced by restriction endonuclease digestion of fulllength cloned DNA sequences, and isolated by electrophoresis on agarosegels. Linkers containing restriction endonuclease cleavage sites can beemployed to insert the desired DNA fragment into an expression vector,or the fragment can be digested at cleavage sites naturally presenttherein. The polymerase chain reaction procedure also can be used toamplify a DNA sequence encoding a desired protein fragment. See, e.g.,PCR Protocols, A Guide to Methods and Applications, Academic Press, NewYork, 1990. Known mutagenesis techniques can be employed to insert astop codon at a desired point, e.g., immediately downstream of the codonfor the last amino acid of the extracellular domain. In anotherapproach, enzymatic treatment (e.g., using Bal 31 exonuclease) can beemployed to delete terminal nucleotides from a DNA fragment to obtain afragment having a particular desired terminus. Linkers that can beligated to the blunt ends produced by Bal 31 digestion and that containrestriction endonuclease cleavage sites are commercially available.Oligonucleotides that reconstruct the amino or carboxy terminus of a DNAfragment to a desired point can be synthesized and ligated to the DNAfragment. The synthesized oligonucleotides include those containing arestriction endonuclease cleavage site upstream of the desired codingsequence and position an initiation codon (ATG) at the amino-terminus ofthe coding sequence.

[0093] Constructs that encode mutant flt3-L polypeptides having variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences in addition tothose affecting flt3-L biological activity, are also included in theinvention. For example, N-glycosylation sites in the flt3-Lextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by the amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro, and Y is Ser or Thr. The human flt3-L protein has twosuch triplets, at amino acids 126-128 and 150-152 of SEQ ID NO:1.Appropriate substitutions, additions or deletions to the nucleotidesequence encoding these triplets result in prevention of attachment ofcarbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846.

[0094] Recombinant expression vectors containing a DNA encoding flt3-Lmutant polypeptide can be prepared using known methods. The expressionvectors include a flt3-L mutant DNA sequence operably linked to suitabletranscriptional or translational regulatory nucleotide sequences, suchas those derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are “operably linked” when theregulatory sequence functionally relates to the flt3-L mutant DNAsequence. Thus, a promoter nucleotide sequence is operably linked to aflt3-L mutant DNA sequence if the promoter nucleotide sequence controlsthe transcription of the flt3-L mutant DNA sequence. An origin ofreplication, or equivalent means for replicating in particular hostcells, and selection genes are used to identify transformants.

[0095] Suitable host cells for expression of flt3-L polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, New York 1985. Cell-freetranslation systems are also appropriate for producing flt3-L mutantpolypeptides using RNAs derived from DNA constructs disclosed herein.

[0096] Suitable prokaryotic hosts include gram negative or gram positiveorganisms, for example, E. coli, Bacillus subtilis, Salmonellatyphimurium, and various other species within the genera Pseudomonas,Streptomyces, Bacillus or Staphylococcus. A mutant flt3-L polypeptidemay include an amino terminal methionine residue to facilitateexpression of the recombinant polypeptide in the prokaryotic host cell.The amino terminal methionine may be cleaved from the expressedrecombinant flt3-L mutant polypeptide.

[0097] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance, and thus provides a simple means foridentifying transformed cells. To construct an expression vector usingpBR322, an appropriate promoter and a flt3-L mutant DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and PGEMI (Promega Biotec, Madison, Wis., USA).

[0098] Promoter sequences used in recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057 (1980); and EP-A- 36776) and tac promoter (Sambrook etal., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1982, at 412). A particularly usefulprokaryotic host cell expression system employs a phage λ_(L) promoterand a c1857ts thermolabile repressor sequence. Plasmid vectors whichincorporate derivatives of the λP_(L) promoter are available from theAmerican Type Culture Collection (ATCC). These vectors include plasmidpHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28(resident in E. coli RRI (ATCC 53082)).

[0099] Flt3-L mutant polypeptides include those expressed in yeast hostcells, preferably from the genus Saccharomyces (e.g., S. cerevisiae).Other genera of yeast, which can be used include Pichia, K. lactis orKluyveromyces. Yeast vectors containing an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene can also beused. Suitable promoter sequences for yeast vectors include, amongothers, promoters for metal-lothionein, 3-phosphoglycerate kinase(Hitzeman et al., J. Biol. Chem. 255:2073 (1980)) or other glycolyticenzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); and Holland etal., Biochem. 17:4900 (1978)), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657; Fleer et. al., Gene, 107:285-195 (1991); and van den Berget. al., Bio/Technology, 8:135-139 (1990). The glucose-repressible ADH2promoter described by Russell et al. (J. Biol. Chem. 258:2674, (1982))and Beier et al. (Nature 300:724, (1982)) is also suitable. Shuttlevectors that can replicate in both yeast and bacteria are constructede.g., by inserting DNA sequences from pBR322 for selection andreplication in, e.g., E. coli (e.g., the Amp^(r) gene and origin ofreplication) into the above-described yeast vectors.

[0100] The yeast α-factor leader sequence directs secretion of mutantflt3-L polypeptides. The α-factor leader sequence can be insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933 (1982); Bitter et al., Proc. Natl. AcadSci. USA 81:5330 (1984) U.S. Pat. No. 4,546,082; and EP 324,274. Otherleader sequences suitable for facilitating secretion of recombinantpolypeptides from yeast hosts are known to those of skill in the art.Modification of a leader sequence near its 3′ end, so that it containsone or more restriction sites, facilitates fusion of the leader sequenceto the structural gene.

[0101] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929 (1978). This protocol selects for Trp⁺ transformants ina selective medium, wherein the selective medium consists of 0.67% yeastnitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20μg/ml uracil.

[0102] Induction of expression in a “rich” medium can be carried out inyeast host cells transformed by vectors containing the ADH2 promotersequence. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenineand 80 μg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0103] Mammalian or insect host cell culture systems can also beemployed to express recombinant flt3-L polypeptides. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin are also used as host cells. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al. Cell 23:175 (1981)), L cells, C127cells, 373 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLacells, and BHK (ATCC CRL 10) cell lines, and the CV-I/EBNA-1 cell linederived from the African green monkey kidney cell line CVI (ATCC CCL 70)as described by McMahan et al., EMBO J. 10:2821 (1991).

[0104] Transcriptional and translational control sequences for usemammalian host cell expression vectors can be excised from viralgenomes. Useful promoter and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, the SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites, can be used to express a structural gene sequence in a mammalianhost cell. Viral early and late promoters are particularly usefulbecause both are easily obtained from a viral genome as a fragment whichmay also contain a viral origin of replication (Fiers et al., Nature273:113 (1978)). Smaller or larger SV40 fragments may also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0105] Exemplary expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama et al. Mol. Cell. Biol. 3:280(1983). A useful system for stable, high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. Mol. Immunol. 23:935 (1986).A useful high expression vector, PMLSV N1/N4, described by Cosman etal., Nature 312:768 (1984) is deposited as ATCC 39890. Additional usefulmammalian expression vectors are described in EP-A- 0367566, and in U.S.patent application Ser. No. 07/701,415, filed May 16, 1991. Vectorsderived from retroviruses are also suitable expression vectors.

[0106] Flt3-L mutant polypeptides include those produced by arecombinant expression system, or purified from cells as naturallyoccurring mutants. One process for producing a mutant flt3-L polypeptidecomprises culturing a host cell transformed with an expression vectorcomprising a DNA sequence that encodes a mutant flt3-L polypeptide underconditions sufficient to promote its expression. Mutant flt3-L is thenrecovered from the culture medium or cell extracts, depending upon theexpression system employed. Procedures for purifying a recombinantprotein vary according to such factors as the type of host cellsemployed and whether or not the recombinant protein is secreted into theculture medium.

[0107] For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Finally, one or more reversed-phasehigh performance liquid chromatography (RP-HPLC) steps employinghydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl orother aliphatic groups) can be employed to further purify mutant flt3-Lpolypeptides. Some or all of the foregoing purification steps, invarious combinations, are well known and can be employed to provide asubstantially homogeneous recombinant protein.

[0108] Mutant flt3-L polypeptides can be affinity purified on columnscomprising the ligand binding domain of flt3. The flt3-L mutantpolypeptides can be removed from an affinity column using conventionaltechniques, e.g., by using a high salt elution buffer or by changing pHor other components depending on the affinity matrix utilized.Alternatively, the affinity column comprises an antibody that binds aflt3-L mutant polypeptide. Monoclonal antibodies directed against mutantflt3-L polypeptides may be derived by methods known to those skilled inthe art.

[0109] Recombinant protein produced in bacterial culture are usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets (for insoluble polypeptides), or from thesupernatant fluid (for soluble polypeptides), followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0110] Transformed yeast host cells can be employed to express flt3-Lmutant polypeptides as secreted polypeptides in order to simplifypurification. Secreted recombinant polypeptide from a yeast host cellfermentation can be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296:171 (1984), which describes twosequential, reversed-phase HPLC steps for purification of a recombinantprotein on a preparative HPLC column.

[0111] Therapeutic Applications of flt3-L Mutant Polypeptides andNucleic Acids

[0112] Flt3-L induces the proliferation and differentiation of cellsexpressing the flt3 receptor. Flt3 has been found to be expressed in thebrain, placenta, tissues of nervous and hematopoietic origin, testis,ovaries, lymph node, spleen, thymus and fetal liver, as well as inleukemias, including acute myelogenous leukemia (AML) and acutelymphocytic leukemias (T-ALL and B-ALL). Flt3-L induces proliferation ofhematopoietic progenitor or stem cells, as well as T cells, early Bcells, and erythrocytes. In addition, flt3-L induces the differentiationof hematopoietic progenitor cells into cell types of different lineages,including dendritic cells, facilitating cells, and NK cells. Flt3-Lmutant polypeptides can therefore be used to treat a variety ofconditions associated with damage to these tissues and cell types.

[0113] Since wild type flt3-L has been shown to stimulate T cellproliferation (see U.S. Pat. No. 5,554,512), flt3-L mutant polypeptidescan be used to treat patients infected with human immunodeficiency virus(HIV). Such treatment includes in vivo administration of mutant flt3-Lpolypeptides, to stimulate proliferation in vivo of CD4⁺ T cells, aswell as ex vivo expansion of isolated T cells. Treatment with flt3-Lmutant polypeptides would elevate or maintain an HIV-infected patient'simmune response. In addition, in vivo treatment would stimulate cells ofthe erythroid lineage, thereby improving a patient's hematocrit andhemoglobin levels.

[0114] Flt3-L mutant polypeptides can be administered either alone or insequential or concurrent combination with cytokines includinginterleukins (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 or IL-15; a colonystimulating factor (CSF) selected from the group consisting of G-CSF,GM-CSF, M-CSF, or GM-CSF/IL-3 fusion proteins; or other growth factorssuch as Stem Cell Factor (SCF), erythropoietin (EPO), leukemiainhibitory factor (LIF), fibroblast growth factor (FGF) orthrombopoietin (TPO).

[0115] The use of flt3-L mutant polypeptides to stimulate production oferythroid cells in vivo for the treatment of anemia is also included inthe invention. Such use comprises administering a flt3-L mutantpolypeptide to a patient in need of such erythroid cell stimulation, inconjunction with or following cytoreductive therapy. The treatment caninclude co-administration of another growth factor, including but notlimited to those listed above.

[0116] The invention also includes the use of flt3-L in peripheral bloodprogenitor or stem cell transplantation procedures. Typically,peripheral blood progenitor cells or stem cells are removed from apatient prior to myelosuppressive cytoreductive therapy, and thenreadministered to the patient concurrent with or following cytoreductivetherapy, to counteract its myelosuppressive effects. The inventionprovides for the use of an effective amount of a mutant flt3-Lpolypeptide in at least one of the following manners: (i) a flt3-Lmutant polypeptide is administered to the patient prior to collection ofthe progenitor or stem cells to increase or mobilize the numbers of suchcirculating cells; (ii) following collection of the patient's progenitoror stem cells, a mutant flt3-L polypeptide is used to expand such cellsex vivo; and (iii) a mutant flt3-L polypeptide is administered to thepatient following transplantation of the collected progenitor or stemcells to facilitate engraftment thereof. The transplantation method ofthe invention can further comprise the use of an effective amount of aanother cytokine, such as those listed above, sequentially orconcurrently with a flt3-L mutant polypeptide. Flt3-L mutantpolypeptides are useful for autologous, syngeneic and allogeneic celltransplantations.

[0117] The invention further includes the use of flt3-L mutantpolypeptides to expand progenitor or stem cells collected from umbilicalcord blood. The expansion may be performed with a flt3-L mutantpolypeptide alone, or sequentially or concurrently with a cytokine fromthe group listed above.

[0118] The term “autologous transplantation” is described in U.S. Pat.No. 5,199,942. Briefly, the term means a method for conductingautologous hematopoietic progenitor or stem cell transplantation,comprising: (1) collecting hematopoietic progenitor cells or stem cellsfrom a patient prior to cytoreductive therapy; (2) expanding thehematopoietic progenitor cells or stem cells ex vivo with flt3-L toprovide a cellular preparation comprising increased numbers ofhematopoietic progenitor cells or stem cells; and (3) administering thecellular preparation to the patient in conjunction with or followingcytoreductive therapy. Progenitor and stem cells may be obtained fromperipheral blood harvest or bone marrow explants. Optionally, one ormore cytokines, selected from the group listed above, can be combinedwith a flt3-L mutant polypeptide to aid in the proliferation ofparticular hematopoietic cell types, or affect the cellular function ofthe resulting proliferated hematopoietic cell population. Of theabove-listed cytokines, SCF, IL-1, IL-3, EPO, TPO, G-CSF, GM-CSF andGM-CSF/IL-3 fusion proteins are preferred, with G-CSF, GM-CSF andGM-CSF/IL-3 fusions being especially preferred. The term “allogeneictransplantation” means a method in which bone marrow or peripheral bloodprogenitor cells or stem cells are removed from a mammal andadministered to a different mammal of the same species. The term“syngeneic transplantation” means bone marrow transplantation betweengenetically identical mammals.

[0119] The transplantation method of the invention described aboveoptionally comprises a preliminary in vivo procedure comprisingadministering a flt3-L polypeptide alone or in sequential or concurrentcombination with a recruitment growth factor to a patient, to recruithematopoietic cells into peripheral blood prior to harvest. Suitablerecruitment factors are listed above, with SCF, IL-1 and IL-3 beingpreferred.

[0120] The method described above optionally comprises a subsequent invivo procedure comprising administering to a patient a flt3-L mutantpolypeptide alone, or in sequential or concurrent combination with anengraftment growth factor to a patient following transplantation of thecellular preparation, to facilitate engraftment and augmentproliferation of engrafted hematopoietic progenitor or stem cells fromthe cellular preparation. Suitable engraftment factors are listed above,with GM-CSF, G-CSF, IL-3, IL-1, TPO, EPO and GM-CSF/IL-3 fusion proteinsbeing preferred.

[0121] Flt3-L mutant polypeptides and nucleic acids can also be used toinduce the differentiation of certain cells in vivo and in vitro. Forexample, large quantities of dendritic cells can be generated from CD34⁺hematopoietic progenitor cells using the flt3-L mutant polypeptides ofthe invention. Following collection of CD34⁺ hematopoietic progenitorsand stem cells, flt3-L mutant polypeptides can be used to expand suchcells in vitro (also known as ex vivo expansion) and to drive such CD34⁺cells to differentiate into dendritic cells of the lymphoid or myeloidlineage. The resulting collection of dendritic cells can be administeredto a patient to provide a stronger and improved immune response to anantigen. Alternatively, the resulting dendritic cells can be used as avaccine adjuvant and can be administered prior to, concurrently with orsubsequent to antigen administration. As vaccine adjuvants, flt3-Lmutant polypeptides can generate large quantities of dendritic cells andother intermediate cells in vivo to more effectively present antigen.The overall response is a stronger and improved immune response and moreeffective immunization to the antigen.

[0122] A procedure for “ex vivo expansion” of hematopoietic stem andprogenitor cells is described in detail in U.S. Pat. No. 5,199,942.Briefly, the method includes the steps of collecting CD34⁺ hematopoieticstem and progenitor cells from a patient from peripheral blood harvestor bone marrow explants and expanding such cells ex vivo. In addition tothe cellular growth factors described in U.S. Pat. No. 5,199,942, otherfactors such as flt3-L, IL-1, IL-3, or c-kit ligand can be used.

[0123] A variety of cell selection techniques are known for identifyingand separating CD34⁺ hematopoietic stem or progenitor cells from apopulation of cells. Methods and materials for identifying and selectingsuch cell types are known. Typically, the first step is to collect bonemarrow or peripheral blood cells using conventional procedures.Peripheral blood progenitor cells (PBPC) and peripheral blood stem cells(PBSC) can be collected using apheresis procedures known in the art.See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616(1994). Briefly, PBPC and PBSC are collected using conventional devices,for example, a Haemonetics Model V50 apheresis device (Haemonetics,Braintree, Mass.). Four hour collections are performed typically no morethan five times weekly until, for example, approximately 6.5×10⁸mononuclear cells (MNC)/kg patient are collected. The cells aresuspended in standard media and then centrifuged to remove red bloodcells and neutrophils. Cells located at the interface between the twophases (i.e., the buffy coat) are withdrawn and resuspended in HBSS. Thesuspended cells are predominantly mononuclear and a substantial portionof the cell mixture are early stem cells.

[0124] Hematopoietic progenitor and stem cells are then isolated fromthe mononuclear cell fraction by any of a variety of procedures known tothose skilled in the art. For example, monoclonal antibodies can be usedto bind to a marker protein or surface antigen protein found on stem orprogenitor cells. Such markers or cell surface antigens forhematopoietic stem cells include flt3, CD34 and Thy-1. Monoclonalantibodies recognizing these antigens have been described. See, e.g.,U.S. Pat. No. 4,714,680 (Anti-My-10). Antibody specific for CD34 iscommercially available from Becton Dickinson, Franklin Lakes, N.J., andanti-Thy-1 monoclonal antibodies can be readily generated using themethods described by Dalchau et al., J. Exp. Med. 149:576 (1979). A flt3receptor binding protein also may be used, such as a monoclonal antibodyspecific for flt3, or the flt3-ligand. The cell binding protein isbrought into contact with the collected cell mixture, and thecombination is allowed to incubate for a period of time sufficient topermit the binding of the desired cell to the cell binding protein.Undesired cells and cell matter are removed, providing a relatively purepopulation of stem cells. Stem or progenitor cells having the CD34marker constitute only about 1% to 3% of the mononuclear cells in thebone marrow. The amount of CD34⁺ stem or progenitor cells in theperipheral blood is approximately 10- to 100-fold less than in bonemarrow.

[0125] Isolation of hematopoietic stem or progenitor cells can beperformed by using, for example, affinity chromatography,antibody-coated magnetic beads, or antibodies fixed to a solid matrix,such as glass beads, flasks, etc. Antibodies that recognize a stem orprogenitor cell surface marker can be fused or conjugated to otherchemical moieties including biotin, which can be removed with an avidinor a streptavidin moiety secured to a solid support, or fluorochromesuseful in fluorescence activated cell sorting (FACS). Preferably,isolation is accomplished by an immunoaffinity column. Immunoaffinitycolumns can take any form, but usually comprise a packed bed reactor.The packed bed in these bioreactors is preferably made of a porousmaterial having a substantially uniform coating of a substrate. Theporous material, which provides a high surface area-to-volume ratio,allows for the cell mixture to flow over a large contact area while notimpeding the flow of cells out of the bed. Typical substrates includeavidin and streptavidin, while other conventional substrates can beused. The substrate should, either by its own properties, or by theaddition of a chemical moiety, display high-affinity for a moiety foundon the cell-binding protein such as a monoclonal antibody. Themonoclonal antibodies recognize a cell surface antigen on the cells tobe separated, and are typically further modified to present a biotinmoiety. It is well known that biotin has a high affinity for avidin, andthe affinity of these substances thereby removably secures themonoclonal antibody to the surface of the packed bed. Such columns arewell known in the art. See Berenson et al., J. Cell Biochem., 10D:239(1986).

[0126] The column is washed with a PBS solution to remove unboundmaterial, and target cells can be released from the beads usingconventional methods. Immunoaffinity columns of the type described abovethat utilize biotinylated anti-CD34 monoclonal antibodies secured to anavidin-coated packed bed are described for example, in PCT Publ. No. WO93/08268. A variation of this method utilizes cell binding proteins,such as the monoclonal antibodies or flt3-L as described above,removably secured to a fixed surface in the isolating means. The boundcell binding protein is then contacted with the collected cell mixtureand allowed to incubate for a period of time sufficient to permitisolation of the desired cells.

[0127] Alternatively, the monoclonal antibodies that recognize the cellsurface antigens can be labeled with a fluorescent label, e.g.,chromophore or fluorophore, and separated by cell sorting according tothe presence of absence or the amount of labeled product.

[0128] An alternative means of selecting quiescent stem cells is toinduce cell death in the dividing, more lineage-committed, cell typesusing an antimetabolite such as 5-fluorouracil (5-FU) or an alkylatingagent such as 4-hydroxycyclophosphamide (4-HC). The non-quiescent cellsare stimulated to proliferate and differentiate by the addition ofgrowth factors that have little or no effect on the stem cells, makingthe non-quiescent cells more vulnerable to the cytotoxic effects of 5-FUor 4-HC. See Berardi et al., Science 267:104 (1995).

[0129] Isolated stem cells can be frozen in a controlled rate freezer(e.g., Cryo-Med, Mt. Clemens, Mich.), and stored in the vapor phase ofliquid nitrogen. Ten percent dimethylsulfoxide can be used as acryoprotectant. After all collections from a donor have been made, thestem cells are thawed and pooled. To induce expansion of the stem cellpopulation in vitro, the cells are incubated in growth medium, such asMcCoy's 5A medium, including 0.3% agar, a flt3-L mutant polypeptide, andoptionally an additional growth factor, e.g., recombinant human GM-CSF,IL-3, and recombinant human GM-CSF/IL-3 fusion molecules (PIXY321), atconcentrations of approximately 200 U/mL, at 37° C. in 5% CO₂ in fullyhumidified air for 14 days. Optionally, human IL-1α or IL-4 may be addedto the cultures. A preferred additional growth factor is IL-3 or aGM-CSF/IL-3 fusion protein.

[0130] Flt3-L mutant polypeptides can also be used to induce hemapoieticcells to differentiate, e.g., into dendritic cells. To inducedifferentiation, collected cells, e.g., CD34⁺ cells, are exposed toeither a flt3-L mutant polypeptide alone or in concurrent or sequentialcombination with one or more of the following cytokines: GM-CSF oranother colony stimulating factor (CSF), erthyopoietin (EPO),thrombopoietin (TPO), Tumor Necrosis Factor α (TNF-α), an interleukin,c-kit ligand or a GM-CSF/IL-3 fusion protein. The CD34⁺ cells are thenallowed to differentiate and commit to cells of the dendritic lineage.The resulting dendritic cells are collected and can either be (a)administered to a patient in order to augment the immune system andT-cell mediated or B-cell mediated immune responses to antigen, (b)exposed to an antigen prior to administration of the dendritic cellsinto a patient, (c) transfected with a gene encoding an antigen-specificpolypeptide, or (d) exposed to an antigen and then allowed to processand present the antigen, ex vivo, to T cells collected from the patientfollowed by administration of the antigen-specific T cells to thepatient.

[0131] The invention allows the use of an effective amount of flt3-Lmutant polypeptide to increase or mobilize dendritic cells in vivo, forexample, in a patient's peripheral blood or other tissue or organs, suchas the spleen. By increasing the quantity of the patient's dendriticcells, such cells may themselves be used to present specific antigen toT cells. For example, the antigen may be one that already exists withinthe patient, such as a tumor, bacterial or viral antigen. Flt3-L mutantpolypeptides may be used, therefore, to boost the patient'slymphocyte-mediated (e.g., T cell or B cell-mediated) ormyeloid-mediated immune response to the already present antigens,resulting in a more effective antigen presentation to the patient'scells. Alternatively, flt3-L mutant polypeptides are administered priorto, concurrently with or subsequent to administration of an antigen to apatient for immunization purposes.

[0132] Flt3-L mutant nucleic acids are also used for gene therapy. Genetherapy procedures include those in which cells transfected withexogenous DNA are administered to a host and allowed to engraft. Seee.g., Boggs, International J. Cell Cloning, 8:80-96 (1990); Kohn et al.,Cancer Invest. 7(2):179-192 (1989); Lehn, Bone Marrow Transpl. 5:287-293(1990); Verma, Scientific American pp. 68-84 (1990). One method oftransferring a gene to a mammal comprises the steps of culturing earlyhematopoietic cells in media comprising a flt3-L mutant polypeptidealone or in sequential or concurrent combination with a cytokineselected from the group listed above; transfecting the cultured cellswith the exogenous gene; and administering the transfected cells to themammal.

[0133] Pharmaceutical Compositions of Flt3-L Mutants

[0134] Pharmaceutical compositions of mutant flt3-L polypeptides areused to treat conditions in which modulation of flt3-L activity isdesirable. Such conditions include myelodysplasia, aplastic anemia, HIVinfection and AIDS, and cancer.

[0135] The pharmaceutical compositions can include growth factors orcytokines in addition to mutant flt3-L peptides or polypeptides. Suchgrowth factors and cytokines include, but are not limited to,interleukins (IL), including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 or IL-15; a colonystimulating factor (CSF) selected from the group consisting of G-CSF,GM-CSF, M-CSF, or GM-CSF/IL-3 fusion proteins; or other growth factorssuch as Stem Cell Factor (SCF), erythropoietin (EPO), thrombopoietin(TPO), leukemia inhibitory factor (LIF) or fibroblast growth factor(FGF). Isolated polypeptides can be further purified by methods known tothose skilled in the art, e.g., HPLC. See, e.g., Fisher, LaboratoryTechniques in Biochemistry and Molecular Biology Work et al., eds.,Elsevier, 1980. Peptides can be synthesized by methods that are known tothose skilled in the art. See, e.g., Solid Phase Peptide Synthesis, 2ded., The Pierce Chemical Company, Rockford, Ill., 1984.

[0136] Pharmaceutical compositions also include nucleic acids encodingmutant flt3-L polypeptides. These nucleic acids are administered in amanner allowing their uptake and expression by cells in vivo.Compositions containing nucleic acids are prepared for administration bymethods that are routine for those skilled in the art.

[0137] Pharmaceutical compositions can include one or more compounds,e.g., nucleic acids, peptides, or polypeptides, and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers arebiologically compatible vehicles, e.g., physiological saline, which aresuitable for administration to a patient.

[0138] Nucleic acids can be administered to a patient by standard vectorand/or gene delivery systems. Suitable gene delivery systems includeliposomes, receptor-mediated delivery systems, naked DNA and viralvectors such as herpes viruses, retroviruses, adenoviruses andadeno-associated viruses.

[0139] Flt3-L mutants can be formulated according to known methods usedto prepare pharmaceutically useful compositions. Flt3-L mutants can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzylalcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,1980. In addition, such compositions can contain mutant fit3-L complexedwith polyethylene glycol (PEG), metal ions, or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, etc., or incorporated into liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of flt3-L mutants. Flt3-L mutants can also be conjugated toantibodies against tissue-specific receptors, ligands or antigens, orcoupled to ligands of tissue-specific receptors. Where flt3 is found onneoplastic cells, flt3-L mutants may be conjugated to a toxin wherebythe flt3-L mutant is used to deliver the toxin to the specific site, ormay be used to sensitize such neoplastic cells to subsequentlyadministered anti-neoplastic agents.

[0140] Flt3-L mutant polypeptides can be administered topically,parenterally, or by inhalation. The term “parenteral” includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. These compositions will typicallycontain a therapeutically effective amount of a flt3-L mutant, alone orin combination with an effective amount of any other active material.Dosages for particular patients depend upon many factors, includingintended use, the patient's size, body surface area, age, the particularsubstance to be administered, time and route of administration, generalhealth and other drugs being administered concurrently. Preliminarydoses can be determined according to animal tests, and the scaling ofdosages for human administration can be performed according toestablished methods. A typical dose of polypeptide, peptide or nucleicacid to be administered to a patient is 100 μg per kilogram of bodyweight.

[0141] Mutant Polypeptides of Flt3-L-Related Growth Factors

[0142] The identification of the key protein residues involved in flt3-Lbinding to flt3 allows the production of novel mutants of related growthfactor polypeptides that can bind to and activate flt3-expressing cells.Such growth factors include Macrophage Stimulating Factor (M-CSF), andStem Cell Factor (SCF). These proteins, which have a high degree ofstructural similarity to flt3-L, can be mutagenized at residuescorresponding to those involved in flt3-L binding to flt3. For example,SCF (also known as c-kit ligand, Steel Factor (SF) and Mast Cell GrowthFactor), is a hematopoietic growth factor that binds the c-kit receptor.See WO 97/38101. Therapeutic use of SCF is hampered by its ability tostimulate mast cells. Mast cell stimulation can result in toxic effectsin vivo, including histamine release and respiratory distress. SeeLyman, Intl. J. Hematol. 62:63-73 (1995). Mutant SCF polypeptides of theinvention maintain activity as hematopoietic growth factors, but do notstimulate mast cells. The invention also includes small molecules whichincorporate key residues for flt3 binding, and which maintain a threedimensional structure that allows binding to and activation offlt3-expressing cells.

EXAMPLES Example 1

[0143] Generation of and Screening for flt3-L Mutants

[0144] The screening assay in which flt3-L mutants were identifiedutilized a flt3-L expression vector, which was subjected to randommutagenesis. Yeast were transformed using the mutagenized vectors, andsubjected to a filter binding assay to detect secreted proteins.Proteins corresponding to flt3-L mutants were detected by reaction withan anti-flt3-L antibody, followed by a flt3-Fc fusion protein, andHRP-goat anti-human IgG, Fcy conjugate, as described below.

[0145] Construction of a Flt3-L Expression Vector

[0146] The flt3-L expression vector was constructed by inserting humanflt3-L nucleic acid into the multicopy expression vector, paADH2. Thesequence of a wild type flt3-L cDNA is disclosed in U.S. Pat. No.5,554,512, and is set forth herein as SEQ ID NO:2. The paADH2 plasmidcontains the 2μ origin of replication and the ADH2 promoter to driveexpression of foreign genes, and the α-factor to direct secretion ofheterologous proteins. Price et al., Gene 55:287-293 (1987). The PIXY456expression vector is derived from paADH2 by addition of a BamH1 siteadjacent to the ASP718 site. Primers JM37(5′-ATTAGGTACCTTTGGATAAAAGACTCAGTGGGACCAGGAC-3′) (SEQ ID NO: 3) andP11673 (5′- ATATGGATCCCTACGGGGCTGTGGCCTCCAGGGGCCG-3′) (SEQ ID NO: 4)were used to amplify human flt3-L from flt3-L clone 9 cDNA (Lyman etal., Blood 83:2795-2801 (1994)). JM37 primer contains an ASP718 site 20bases from the 3′ end of the α-factor, and fuses the α-factor leader tothe flt3-L PCR product. P11673 contains a BamH1 site for cloning intoPIXY456. The amplified flt3-L fragment was digested with Asp718 andBamH1 and ligated into PIXY456 to construct PIXY771. The NFSglycosylation site at positions 123-125 of the flt3-L gene was mutatedto NFA using primer JM40(5′-CCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCGCCCGGTGCCTGG-3′)(SEQ ID NO: 5). Primers JM40 and P11673 were used to amplify the 3′ endof the human flt3-L gene. This amplified fragment was digested with Bsaland BamH1 and used to replace the Basl-BamH1 fragment in PIXY771,resulting in plasmid PIXY797. All constructs were confirmed by DNAsequence analysis.

[0147] Mutagenesis and Transformation

[0148] The flt3-L expression vector was subjected to random mutagenesisand used to transform yeast. Mutagenic PCR was performed as previouslydescribed (Leung et al., Technique 1:11-15 (1989)). DNA sequenceanalysis identified 100 point mutations, including silent mutations,from 59 clones. The flt3-L nucleotide lesions obtained includedtransitions (49 A/T→G/C, 18 G/C→A/T) and transversions (21 A/T→T/A,3A/T→C/G, 7 G/C→T/A, 2 G/C→C/G).

[0149] Yeast transformations were performed by homologous recombination(Muhlard et al., Yeast 8:79-82 (1992)). In these transformations,gel-purified gapped vector DNA and the flt3-L PCR fragment were added toLiOAc-treated yeast. The flt3-L PCR product contains 30 bp 5′ to and 60bp 3′ to the wild type flt3-L gene. These two regions are homologous tothe regions immediately adjacent to the gap in the vector DNA.Transformed yeast were plated on YNB (-trp) selective medium (Sherman,Methods Enzymol. 194:3-21 (1991)) and grown at 30° C. until individualcolonies were 0.5 to 2 mm in diameter, about 2 to 3 days.

[0150] Screening Assay to Detect Flt3-L Mutants

[0151] The transformed yeast colonies were then used in the screeningassay, as described below. Colonies of 0.5 to 2 mm in diameter werelifted onto cellulose acetate membranes. A second “capture” membrane,made of nitrocellulose, was coated with 0.1 mg/ml M5α-flt3-L, amonoclonal antibody that specifically binds flt3-L, in 50 mM phosphate,pH 7.4, 150 mM NaCl (PBS) for 16 hours at 4° C. M5α-flt3-L is anon-neutralizing antibody that binds flt3-L without interfering with theflt3-flt3-L binding interaction. Progressive deletions of flt3-Lindicate that residues carboxy-terminal to cys-131 are dispensable fornormal activity, but are required for binding to M5α-flt3-L. Lyman etal., Blood 86:4091-4096 (1995).

[0152] The capture membrane was subsequently blocked for four hours at4° C. in blocking solution (PBS containing 3% (w/v) BSA, 5% (w/v) nonfatdry milk), then washed twice in PBS. The wet capture membrane was thenplaced on a YEPD plate (Sherman, Methods Enzymol. 194:3-21 (1991)),taking care to avoid air pockets. The capture membrane was then coveredwith the cellulose acetate membrane with the yeast colonies uppermost,again taking care to avoid air pockets. The membrane sandwich was markedby needle puncture for orientation, and the assembly was incubated at30° C. for 18 to 22 hours. The cellulose acetate membrane with colonieswas removed and placed on a fresh YEPD plate (Sherman, Methods Enzymol.194:3-21 (1991)) and stored at 4° C. for later recovery of the yeast.

[0153] The capture membrane was then used to detect flt3 binding to theimmobilized flt3-L mutant proteins. This protocol was carried out atroom temperature and all reagents and washes were in PBS containing 1%(w/v) BSA, unless otherwise stipulated. The capture membrane was blockedfor one hour in blocking solution. After washing three times, themembrane was probed for one hour with 0.5 μg/ml human flt3-Fc. Thissoluble form of the human flt3 protein was constructed using a protocolpreviously used to make a soluble form of the murine flt3 protein(described in Lyman et al., Cell 75:1157-1167 (1993)). Human flt3nucleic acid was amplified by PCR and cloned, as previously described(Rosnet et al., Blood 82:1110-1119 (1993)). In the flt3-Fc polypeptide,the entire extracellular domain of the human flt3 (ending at the aminoacid just before the start of the transmembrane region) is fused to theFc portion of human immunoglobulin G (IgG). Flt3-Fc fusion proteinexpressed in 293/EBNA cells was purified using protein A-Sepharose(Pharmacia).

[0154] After being probed with flt3-Fc, the membrane was washed threetimes over 10 minutes, then probed for one hour with 0.53 μg/ml HRP-goatanti-human IgG, Fcγ conjugate (Jackson Immuno Research Laboratories).The membrane was then washed three times over 30 minutes in PBS, anddeveloped with 4-chloro-1-naphthol (BioRad) in PBS plus 18% methanol and0.002% hydrogen peroxide. The enzymatic reaction was typically stoppedafter 10 to 15 minutes by washing in distilled water, followed bydrying.

[0155] Single yeast colonies whose receptor binding signal differed fromthe wild type background were isolated and plated on selective medium.Colonies were identified and isolated by alignment of the two membranesvia previously made needle puncture marks. Two ml YEPD cultures of thechosen colonies were grown for three days at 30° C., after which thecells and supernatant were separated by centrifugation (12,000 rpm,Beckman microfuge, 5 minutes, 22° C.). Cells were stored at −20° C. forlater retrieval of plasmid DNA for sequencing and subcloning.Supernatants were used immediately for subsequent screens, or stored at4° C.

Example 2

[0156] Characterization of Flt3-L Mutant Polypeptides

[0157] Identification of Flt3-L Mutant Polypeptides

[0158] The receptor-binding properties of the secreted flt3-L mutantswere assessed visually by noting the variation from wild type flt3-L inintensity of the stained spots after the enzyme-linked reaction. Mostcolonies secreted flt3-L which stained similarly to wild type. About 1%of the colonies, however, gave rise to colonies devoid of stain. Theseclear white colonies were designated as receptor-binding deficient(flt3-L-), and were isolated by reference to their positions on thecellulose acetate membrane. The appearance of stained spots whoseintensity was greater than that of wild type was a far more rare event.These dark colonies were isolated and designated flt3-L⁺⁺, to indicateincreased receptor binding properties over wild type. Approximately60,000 colonies were screened, with up to 300 colonies assayed perstandard size petri dish (82 mm diameter). All of the detectableflt3-L⁺⁺colonies (114 total) were isolated, along with 214 flt3-L⁻colonies, which represented only a portion of the flt3-L⁻ species.

[0159] Electrophoresis and Western Blotting

[0160] Supernatants from all of the flt3-L⁻ and flt3-L⁺⁺ yeast cultureswere analyzed by Western blot, to assess the amount and quality ofmutant flt3-L protein being secreted by individual yeast colonies. Thebuffers, stains, polyacrylamide gels and PVDF membrane used forelectrophoresis were purchased from Novex, and electrophoresis wasperformed as described by the manufacturer. For Western blot analysis,yeast supernatants were run on 4-20% SDS-polyacrylamide gels and thentransferred to PVDF membranes. The blots were probed with the M5α-flt3-Lmonoclonal antibody (Lyman et al., Blood 86:4091-4096 (1995)), followedby HRP-goat anti-rat IgG conjugate (Zymed) and visualized with4-chloro-1-naphthol reagent (BioRad), according to manufacturer'sinstructions.

[0161] Mutants with low levels of expression or grossly alteredstructures were eliminated from further study. Of the 214 flt3-L⁻colonies, 94 were discarded because little or no protein was secreted,five were discarded because only high molecular weight smears wereobserved on Western blot, possibly due to hyperglycosylation, and anadditional four were discarded due to lower than expected molecularweight. The specific activity of the remaining flt3-L mutant proteinswas determined by subjecting supernatant from the yeast coloniessecreting the mutant proteins to ELISA and WWF7 cell proliferationassays. These assays to measure the level of expression of a particularflt3-L mutant polypeptide, and its biological activity, respectively.The ELISA assay has been previously described (Lyman et al., Oncogene10:147-149 (1995)). Biological activity, as tested in the murine WWF7cell assay, was assessed using [³H]thymidine incorporation assays,according to previously published protocols (Brasel et al., Leukemia9:1212-1218 (1995)). Species with low levels of protein expression orspecific activities near that of wild type were discarded. The remaining30 flt3-L⁻ species were subjected to DNA sequence analysis. A similarprocess was used to select 29 flt3-L⁺⁺ species for DNA sequenceanalysis.

[0162] For sequence analysis, plasmid DNA was rescued from yeast clonesand sequenced with synthetic primers that hybridize to the coding andnoncoding strands of the vector DNA 5′ and 3′ the flt3-L gene. Theresults are shown in Table 1. The flt3-L mutant polypeptides aredesignated by a first letter, followed by a number, followed by a secondletter. The first letter is the one-letter abbreviation for an aminoacid found in the mature human flt3-L polypeptide. The number in thedesignation is the position of that amino acid in mature human wild typehuman flt3-L (SEQ ID NO:18). The second letter in the designation is theone-letter abbreviation for the amino acid found at that position in themutant flt3-L polypeptide. For example, D3G refers to a mutant in whichGlycine (G) has been substituted for the Aspartic Acid (D) at position 3of the mature wild type flt3-L protein (SEQ ID NO:18).

[0163] Since amino acids 1-26 of the full length flt3-L protein (SEQ IDNO:1) are cleaved to produce the mature protein, the first amino acid ofthe mature wild type human flt3-L polypeptide (SEQ ID NO:18) is thethreonine at position 27 of the full length polypeptide (SEQ ID NO:1). Anegative number in a designation, e.g., −3 in L-3H, refers to the -3position of the mature human flt3-L wild type polypeptide, i.e.,position 24 of the full length flt3-L polypeptide (SEQ ID NO:1). In thesequence listing, for mutations occurring within the mature flt3-Lpolypeptide, the sequences begin at the threonine at position 27 of thefull length protein (SEQ ID NO:1). For mutations occurring outside themature protein, e.g., L-3H, the position of the mutation is listed asposition 1 in the sequence listing. For example, in L-3H, position 1 inthe sequence listing corresponds to position 24 in the full length humanflt3-L polypeptide (SEQ ID NO:1).

[0164] Table 1 is divided into two sections, one showing species thatwere designated as deficient in receptor binding (flt3-L⁻), and theother showing species that were designated having increased receptorbinding (flt3-L⁺⁺), according to the results of the initial filterbinding assay. Multiple amino acid substitutions occurred in 13 of the59 flt3-L DNA sequences. Flt3-L⁻ clones with multiple amino acidsubstitutions included: L-3F/R95C; T1A/R55L/F96V; D3G/F15Y;D3G/I11F/V113E; P10S/M57V/T62S; I11Y/A35P/F87L; I11Y/L139Q; S13P/M68V;S36P/N37D/L148Q; L96F/S136T. In a number of cases, single mutations weresubcloned from flt3-L mutants that had sustained multiple mutations.Subcloning was carried out by inserting various flt3-L mutants into theE. coli vector, pocus-1 (Novagen). The desired flt3-L mutant, having asingle mutation, was generated by DNA restriction, fragment isolation,and ligation into appropriate vectors. This procedure allowed theassignment of function to individual residues for some of themultiply-substituted proteins. TABLE I Characteristics of Flt3-LMutants: Yeast Medium FL:M/SCF: H-M/M- ELISA^(c) Sp.Ac. MUT^(d)Independent^(f) Mutant_(a) CSF:H-Mb (μg/ml) Sp.Ac. WT (n)^(e) Isolate, nWild Type 5.79   1.00^(g)− (11)  NA Flt3-L − D3G D/NA/S-S 3.98 0.81 ±0.09 (2)  1^(h) H8R H/R-G/H-H 5.01 0.00 (3) 1 S9G S/N-N/M-M 2.69 0.13 ±0.06 (3) 1 P10S P/R-P/I-I 1.83 0.00 (3)  1^(h) I11Y I/V-V/G-G 3.06 0.00(3)  1^(h) S13P S/N-D/G-G 5.71 0.00 (4) 2 S13F S/N-D/G-G 1.83 0.00 (2) 1F15L F/V-V/L-L 3.49 0.00 (2) 1 F15Y F/V-V/L-L 2.26 0.19 ± 0.04 (3) 1^(h) R20C R/K-K/R-Q 2.02 0.11 ± 0.09 (2) 1 R55C R/D-L/D-D 2.51 0.05 ±0.03 (3) 3 R55L R/D-L/D-D 1.58 0.29 ± 0.02 (2)  1^(h) A64T A/F-F/R-K2.46 0.09 ± 0.06 (2) 1 V75A V/L-L/L-L 2.65 0.18 ± 0.07 (2) 1 F81LF/D-D/R-N 1.90 0.06 ± 0.03 (2) 2 F81S F/D-D/R-N 2.71 0.00 (3) 1 F87LL/K-E/T-T 1.22 0.01 ± 0.02 (2) 1 P90S L/S-A/Y-Y 1.92 0.09 ± 0.06 (2) 1R95C R/E-E/R-R 1.40 0.26 ± 0.01 (2)  1^(h) V113E L/D-D/N-N 5.13 0.80 ±0.11 (2)  1^(h) K116E K/K-K/K-K 6.54 0.02 ± 0.01 (2) 1 F124L F/T-T/S-T2.22 0.22 ± 0.06 (5) 2 F124S F/T-T/S-T 2.22 0.00 (4) 1 Flt3-L++ L-3H N/A6.40 1.86 ± 0.32 (3) 2 H8Y H/R-G/H-H 6.07 1.71 ± 1.04 (3) 1 L26FL/P-P/M-M 6.59 0.69 ± 0.17 (3) 1 L27P L/.-./E-E 13.91 0.07 ± 0.03 (4) 2V34L V/L-L/F-F 3.79 0.58 ± 0.15 (3) 1 K84E S/E-L/S-S 7.34 2.88 ± 0.62(21)  14  K84T S/E-L/S-S 4.82 1.63 ± 0.29 (2) 1 W118R C/F-F/L-L 4.492.19 ± 0.19 (2) 1 Q122R Q/S-S/I-I 4.39 2.16 ± 0.25 (5) 1 # aredesignated as flt3 binding deficient (flt3-L⁻), while the last 9 species(i.e., L-3H to Q122R) are designated as having increased flt3 binding(flt3-L⁺⁺), according to the results of the initial filter bindingassay.

[0165]^(a)Mutants are named using a first letter, followed by a number,followed by a second letter. The first letter is the one-letterabbreviation for a wild type amino acid; the number is the position ofthat amino acid in wild type mature flt3-L (i.e., flt3-L from whichamino acids 1-26 of SEQ ID NO:1 have been removed); and the secondletter is the one-letter abbreviation for the replacement amino acid.The first 23 species (i.e., D3G to F124S), are designated as flt3binding deficient (flt3-L⁻), while the last 9 species (i.e., L-3H toQ122R) are designated as having increased flt3 binding (flt3-L⁺⁺),according to the results of the initial filter binding assay.

[0166]^(b)The column labeled FL:M/CSF:H-M/M-CSF:H-M refers to thecorresponding murine flt3-L (FL:M), human and murine SCF (SCF:H-M) andhuman and murine M-CSF (M-CSF:H-M) amino acid residues, based on thesequence alignment of native human and murine flt3-L, SCF and M-CSFproteins (Hannum et al., Nature 368:643-648 (1994)).

[0167]^(c)The level of flt3-L expression as determined by ELISA of yeastsupernatants.

[0168]^(d)Direct assay of yeast supernatants yielded the ratio of cellproliferation activity in WWF7 cells (units/ml) to concentration offlt3-L protein as determined by ELISA (ng/ml). The ratio of specificactivity of mutant to wild type flt3-L in a given assay is averaged overone or more independent assays. A value of zero indicates theproliferation activity of the mutant flt3-L protein was below the limitof detection of the assay.

[0169]^(e)The number of independent assays is represented by n.

[0170]^(f)Independent isolate, n, refers to the number of times aparticular mutation was isolated. NA stands for “not applicable”.

[0171]^(g)The specific activity of WT protein averaged over 11independent assays equals 0.95±0.34 units/ng.

[0172]^(h)These mutants were subcloned from species that had multipleamino acid substitutions.

[0173] The flt3-L mutants isolated and sequenced are shown in Table 1,along with their levels of expression, as determined by ELISA, andspecific activities, as determined by the ratio of cell proliferationactivity in WWF cells (units/ml) to concentration of flt3-L protein asdetermined by ELISA. As shown in Table 1, thirty two different aminoacid substitutions that alter flt3-L biological activity were found at24 sites. FIG. 2 is a linear representation of flt3-L amino acidsubstitutions in the mutants identified in this study. The top of thefigure shows the secondary structure of the wild type human flt3-Lprotein, including the intramolecular disulfide linkage, placement ofβ-sheet segments, and placement of α-helices A through D based onsequence alignment with the M-CSF protein (Hannum et al. Nature368:643-648 (1994)). The bottom of the figure shows the primarystructure of wild type flt3-L, superimposed with the relative specificactivity profile of mutants with single amino acid substitutions.Positions at which more than one substituting amino acid were found arerepresented by the substitution that induced the greatest perturbationin relative specific activity.

[0174] As shown in FIG. 2 at bottom, when the collection of 24 positionsalong the primary structure of flt3-L is plotted, three linear clustersof high frequency amino acid substitution, or mutational “hot spots,”appear at positions 8-15, 81-87 and 116-124 of the mature wild typeflt3-L polypeptide. Each of the three “hot spots” contains, at positions8, 84, 118 and 122 of the mature wild type protein, respectively, aminoacid substitutions that improve the biological activity of flt3-L.Mapping of these hot spots onto a structural model of flt3-L indicatesthat mutations on the solvent-exposed surface of the N terminus of helixA, or disruption of the packing of helix A, is deleterious to thefunction of flt3-L, as indicated by the large number of mutationsisolated between positions 8 through 15 of the mature flt3-L protein.

[0175] As indicated in Table 1, while some of the flt3 mutantpolypeptides contain substitutions at the same position, thesesubstitutions may be by a different amino acid in different mutantpolypeptides. For example, in F15Y, phenylalanine is replaced at the 15position of the mature wild type flt3-L polypeptide by tyrosine, whilein F15L, phenylalanine is replaced at this position by leucine. Theaddition of the single hydroxyl group from the tyrosine side chain inF15Y reduces its activity by 80%, as measured in the WWF7 assay, whilethe conservative substitution in F15L completely abolishes detectableactivity in this assay. The K84E (SEQ ID NO:14) substitution wasdetected in 14 flt3-L⁺⁺ independent isolates, and one flt3-L⁺⁺ K84T (SEQID NO:15) substitution mutant was obtained. In two cases, mutants with aflt3-L⁺⁺ phenotype had an amino acid substitution at the −3 position ofmature flt3-L, i.e., a position outside the mature protein. The flt3-Lused contains an additional three residues, derived from the signalsequence, which are amino terminal to the threonine that is the firstresidue of the mature flt3-L protein (SEQ ID NO:18). Amino terminalsequence analysis of wild type protein has confirmed this conclusion.Thus, a substitution outside the mature protein, i.e., the substitutionof leucine by histidine at the −3 position, is able to increase flt3-Lbiological activity. The screen also produced three mutants, R20C, R55Cand R95C, that have substitutions introducing cysteine residues.

[0176] The L27P mutation (SEQ ID NO:13) occurs at the putative carboxyterminus of helix A of wild type flt3-L, i.e., at the proposeddimerization interface in the model for flt3-L quaternary structure.L27P was isolated in the primary membrane screen as a flt3-L⁺⁺ species,but on secondary screening it was flt3-L⁻. While L27P was isolatedindependently twice as a flt3-L⁺⁺ species, its activity is only 7-20% ofwild type (see FIG. 1 and Table 1). Since L27P is expressed at almostthree times the level of wild type protein, this high level ofexpression likely accounts for the flt3-L⁺⁺ signal observed in theprimary membrane screen. Other mutations identified in the screen thatmap to the dimerization interface are L26F (SEQ ID NO:12) and A64T (SEQID NO:9).

Example 3

[0177] Purification and Analysis of Flt3-L Mutant Polypeptides

[0178] Flt3-L mutants exhibiting greater than wild type specificactivity in the WWF7 assay, as shown in Table 1 (e.g., H8Y (SEQ IDNO:11), K84E (SEQ ID NO:14), K84T (SEQ ID NO:15), W118R (SEQ ID NO:16),and Q122R (SEQ ID NO:17) ), were purified. Mutants with specificactivities lower than wild type (e.g., H8R, I11Y, F81S, K116E) wereselected for further study based on the relative proximity of theirsubstitutions to mutations resulting in a flt3-L⁺⁺ phenotype, and nearwild type levels of expression. The L27P (SEQ ID NO:13) mutant, which ispredicted to disrupt the dimerization interface of flt3-L, was alsopurified.

[0179] Preparation of Recombinant Human Flt3-L

[0180] Wild type and mutant flt3-L proteins were purified to greaterthan 90% homogeneity, according to the following protocol. Yeast medium(1.2 L) was filtered through a 0.22 μm membrane. The pH of the mediumwas adjusted to 4.0 by the addition of glacial acetic acid with rapidmixing, and filtered through a 0.22 μm membrane a second time(conductivity 2-6 mOhms). The filtrate was applied to a 30 ml FractogelEMD SO3-650 (M) (EM Separations), equilibrated with 25 mM NaCH₃COO/50 mMNaCl, pH 4.0 at 20 ml/min. Protein was eluted with 25 nM MES/200 mMNaCl, pH 6.0. The pH was adjusted to 7.5-8 by the addition of {fraction(1/20)}th volume of 1 M Tris, pH 9.0, and the solution was then filteredthrough a YM100 membrane (Amicon).

[0181] Flt3-L was affinity purified by passing the filtrate over acolumn of the monoclonal antibody M5α-flt3-L conjugated toCNBr-activated Sepharose 4B (Pharmacia), equilibrated in 50 mMNaHPO₄/300 mM NaCl, pH 7.4. Protein was eluted with 25 mM NaHPO₄, pH11.3, and the eluant was neutralized by addition of {fraction (1/100)}thvolume 1 M monobasic phosphate. The eluant was concentrated and appliedto a Superdex 200 column (Pharmacia) equilibrated in PBS at 2.5 ml/min.The dimer peak was collected, concentrated, filtered through a 0.22 μmmembrane and stored at 4° C. The concentration of purified flt3-Lprotein was determined by duplicate quantitative amino acid analysisafter acid hydrolysis, and the results averaged. The level ofcontamination of purified protein was assessed visually by running 2-4μg of various flt3-L proteins on 16% SDS-polyacrylamide gels and stainedwith colloidal Coomassie stain. In all cases, the proteins were detectedas a single species (>90%) with a Mr of 18,000.

[0182] The amino acid sequence of the flt3-L protein used to name themutants identified herein is numbered according to the mature fullyprocessed soluble protein, having the amino terminal residuesThr-Gln-Asp at positions 1-2-3, respectively (i.e., positions 27, 28 and29 of full length wild type flt3-L (SEQ ID NO:1)), (see Lyman et al,1994). The gene construct utilized for expression in yeast encodes anadditional three amino acids (Leu-Ser-Gly) amino terminal to the matureprotein. Amino terminal sequence analysis of purified yeast-derivedflt3-L protein shows that 80% of the amino terminal sequence isLeu:Ser:Gly:Thr:Gln:Asp, while 20% begins at the penultimate residue,Serine. The specific activity and K_(di) for receptor binding of thisyeast-derived protein (K_(di)=0.09 nM) used in this study is similar toflt3-L having native mature sequence (K_(di)=0.08 nM), and expressed inmammalian cells, where K_(di) is defined as the dissociation constant ofunlabelled flt3-L for flt3, as determined by inhibition of binding of¹²⁵I labelled flt3-L. Therefore, this yeast-derived protein is referredto as wild type flt3-L.

[0183] Radiolabeling, Binding Assays, and Data Analysis

[0184] Purified recombinant flt3-L was labeled with ¹²⁵I using a solidphase chloramine T analog (Iodogen, Pierce Chemical, Rockford, Ill.) toa specific radioactivity of 4×10¹⁴ cpm/mmol with no detectable loss ofspecific binding activity as assessed by inhibition assays withunlabeled flt3-L.

[0185] Binding assays were performed using a phthalate oil separationmethod as described previously for murine ¹²⁵I-GM-CSF (Park et al., J.Biol. Chem. 261:4177-4183 (1986)). Briefly, BAF-BO3 cells transfectedwith human flt3 cDNA (0.5-1×10⁶) were incubated with serial dilutions of¹²⁵I-flt3-L in binding medium (RPMI 1640, 2.5% bovine serum albumin,0.2% NaN3, 20 mM Hepes, pH 7.2) in 96 well microtiter plates maintainedon a mini-orbital shaker (Bellco) at 37° C. for 90 minutes. Inhibitionbinding assays were carried out by holding the radiolabeled flt3-Lconcentration constant at 0.3 nM, while the concentration of unlabeledcompetitor proteins ranged from 150 nM to 0.001 nM. The ratio of K_(di)of mutant to wild type flt3-L, determined in at least duplicate, in agiven assay was averaged over at least three independent assays and thestandard error of the mean (SEM) reported. The data are shown in FIG. 3.These data were generated using the BAF/hflt3 cell proliferation assay.For this assay, cells expressing human flt3 (BAF/hflt3 cells) wereconstructed from BAF/B03 cells, using a procedure previously describedfor producing BAF/B03 cells expressing the murine flt3 (Lyman et al.,Cell 75:1157-1167 (1993)). Expression of human flt3 by BAF/hflt3 cellswas confirmed by examining the capacity of the cells to proliferate inresponse to soluble flt3-L, and by flow cytometric analysis usingbiotinylated flt3-L.

[0186] The values of flt3 binding and BAF/hflt3 cell proliferativeactivity by purified flt3-L mutants are shown in FIG. 3. The ordinate isthe Log¹⁰ of the value relative to wild type flt3-L, and the abscissa isthe flt3-L mutant designation. Shown are K_(ai) mutant/K_(ai) wild type(filled bars) and specific activity mutantispecific activity of wildtype (open bars). The K_(d) and K_(di) for wild type protein is0.13±0.04 nM (SD) and 0.09±0.02 nM (SEM), respectively (n=7). The ratioof K_(ai) mutant to wild type flt3-L, determined in at least duplicate,in a given assay is averaged over at least three independent assays andthe standard error of the mean (SEM) is reported.

[0187] Specific activity of purified mutant or wild type protein wascalculated as the ratio of proliferation activity in BAF/hflt3 cells(units/ml) to concentration of flt3-L protein as determined by aminoacid composition (ng/ml). The ratio of specific activity of mutant towild type flt3-L, determined in duplicate, in a given assay was averagedover at least three independent assays and the standard error of themean (SEM) is reported. ND stands for “not determined.” Usingradiolabeled yeast-derived protein, a k_(di) of 0.08±0.01 nM forrecombinant CHO-derived human flt3-L, which has native human sequence,was obtained.

[0188] Mutations that increase biological activity relative to wildtype, i.e., H8Y (SEQ ID NO:11), K84E (SEQ ID NO:14), K84T (SEQ IDNO:15), W118R (SEQ ID NO:16) and Q122R (SEQ ID NO:17), were identifiedin each of the three flt3-L mutational “hot spots” identified in thisstudy. Two mutations were isolated in which histidine at position 8 inthe mature flt3-L polypeptide was substituted, i.e., H8R, which isflt3-L⁻, and H8Y, which is flt3-L⁺⁺. This histidine is conserved inmurine flt3-L and the related cytokine, M-CSF. When the flt3-L and M-CSFprotein sequences are aligned, his-8 of the mature flt3-L polypeptide isequivalent to his-9 of M-CSF.

Example 4

[0189] Physical Characterization of flt3-L Mutants

[0190] The results of physical characterization tests indicate that theisolated flt3-L mutant polypeptides maintain the structure of the nativeflt3-L polypeptide. As detailed below, flt3-L mutants were found tomaintain the dimeric structure and the helical content of wild typeflt3-L.

[0191] Determination of Stokes Radius in Purified Flt3-L Mutants

[0192] Stokes radius was measured in purified flt3-L mutants in thefollowing manner. Gel filtration chromatography of purified proteins wasperformed on a 300×7.8 mm Bio-Sil 125-5 column (RioRad) in PBS at a flowrate of 1 ml/min. The elution profiles were monitored at an absorbancewavelength of 280 nm. The proteins were loaded on the column at 45 μginjections at a concentration of 0.1 mg/ml. Standard globular proteinswere loaded at a concentration of 0.1 mg/ml and included thyroglobulin,gamma globulin, bovine serum albumin, ovalbumin, carbonic anhydrase,myoglobin, and cytochrome C (MW=670, 158, 66, 44, 29, 17, 12.4 kDa,respectively). A calibration curve was constructed by plotting log M vsK_(sec), where K_(sec)=(Ve−Vo)/Vt−Vo), where Vo is the void volume asdetermined by thyroglobulin; Vt, the total volume as determined by NaN₃;and Ve, the elution volume of the target protein. The Stokes radius ofpurified flt3-L was determined in duplicate and the mean value isreported in Table 2.

[0193] Circular Dichroism Analysis of Flt3-L Mutant Polypeptides

[0194] Circular dichromism spectra of proteins were obtained using aJasco 500 c spectropolarimeter interfaced with an IBM AT computer. Rawdata (ellipticities) were processed, after averaging and correction forappropriate solvent blanks, according to the equation: MRE=[θ_(obs))(MRW)]/101c, where MRE is the mean residue ellipticity in (deg)(cm²)/dmol, θ_(obs) is the observed ellipticity in mdeg, and MRW is themean residue weight, 1 is the cell pathlength in cm, and c is theconcentration of protein g/ml. All spectra were measured in a 0.1 mmcell in PBS at 22° C., between 260-195 nm using a 1 nm bandwidth and a 1second time constant. The percent helical content was estimated aspreviously described (Sreerama et al., Anal. Biochem. 209:32-44 (1993)).Results are shown in Table 2. TABLE 2 Physical Characteristics of Flt3-LMutants: Purified Protein Stokes Percent Mutant Radius (x10³) Helix WT40.8 50 H8R 40.2 50 H8Y 40.8 51 I11Y 41.1 50 L27P 38.1 50 F81S 41.1 50K84E 43.5 49 K84T ND 53 K116E 45.6 49 W118R 39.4 43 Q122R 38.5 51

[0195] With the exception of W118R, none of the mutants analyzed showedgross structural perturbations. Therefore, most of mutations identifiedare likely in positions of flt3-L that are directly involved in theenergetics of receptor binding.

[0196] Monomeric unglycosylated flt3-L has a molecular weight of 17,686daltons. When analyzed by SDS gel electrophoresis, yeast-produced flt3-Lmigrates at a mass of approximately 21,000 daltons, due to the presenceof core glycosylation at a single N-linked site. Stokes radiusmeasurements, as determined by size exclusion chromatography(Mr=40,000), indicate that both the wild type and mutant proteins aredimeric. In addition, the helical content of the wild type and mutantproteins, as determined by their circular dichroism spectra, aresimilar.

[0197] The purified mutants were also subjected to native gelelectrophoresis. In all cases, the mobility of the mutant proteinsrelative to the mobility of wild type protein corresponded to the chargedifferences associated with the particular amino acid substitution inthe mutant protein, confirming that the assessment of substitutions inthese mutants is correct.

[0198] The helical content of the flt3L mutant polypeptide containingthe W118R substitution is 7% less than that of wild type protein. Ahydrophobic residue occupies the equivalent site of position 118 of themature flt3-L polypeptides in murine and human SCF (phenylalanine), andin murine and human M-CSF (leucine), as determined by amino acidsequence alignment. Hannum et al., Nature 368:643-648 (1994). In W118R,this hydrophobic residue has been replaced with arginine, a basicresidue. Substitution with this basic residue results in a mutant withincreased flt3-L biological activity, despite the fact that thismutation disrupts the helical content of flt3-L.

Example 5

[0199] Construction and Analysis of Flt3-L Multiple Mutant Polypeptides

[0200] Several mutant flt3-L polypeptides containing more than onesubstitution conferring a flt3-L⁺⁺ phenotype were constructed.Characteristics of two of these multiple mutant flt3-L polypeptides areshown in Table 3. A mutant containing both the K84E and Q122Rsubstitutions, constructed by subcloning gene fragments, was expressedat near wild type levels. WWF7 cell proliferation assays of supernatantsfrom yeast expressing the K84E/Q122R mutant polypeptide indicated thatthe total biological activity was equal to the sum of the activities ofthe K84E and Q122R mutants.

[0201] A quadruply substituted flt3-L mutant, L-3H/H8Y/K84E/Q122R,containing the L-3H, H8Y, K84E, and Q122R substitutions, was constructedby PCR mutagenesis, expressed and purified. The 5′ oligo JM116.46(5′-TGGATAAAAGAcacAGTGGGACCCAGGACTGCTCCTTCCAATAcag-3′) (SEQ ID NO: 6),encoding the L-3H and H8Y substitutions (substituted codons are in lowercase), and a 3′ vector primer were used to amplify the double mutantK84E/Q122R. A second PCR reaction was used to extend the 5′ end usingthe oligo JM117.42(5′-TGGATAAAAGACACAGTGGGACCCAGGACTGCTCCTTCCAATACAG-3′) (SEQ ID NO: 7)and 3′ vector primer. The PCR product was introduced into the PIXY456expression vector by recombination as described above. All constructswere confirmed by DNA sequence analysis. As shown in Table 3, receptoraffinity for the purified L-3H/H8Y/K84E/Q122R mutant is over eight timesgreater than wild type protein, and cell proliferation activity is threetimes greater. TABLE 3 Binding and Biological Characteristics ofFlt3-L⁺⁺ Multiple Mutants Sp.Ac. MUT K_(ai) MUT Mutant Sp.Ac. Wt^(a)K_(ai) Wt^(b) WT 1.0 1.0 H8Y 2.1 ± 0.1 2.8 ± 0.1 K84E/Q122R 2.9 ± 0.73.4 ± 1.6 L-3H/H8Y/K84E/Q122R 3.0 ± 1.1 8.6 ± 0.8

Example 6

[0202] Modeling of Flt3-L

[0203] A model of the quaternary flt3-L structure was generated usingFOLDER, a distance geometry-based method for homology modeling.Srinivasun et al., Protein Sci. 2:277-289 (1993). FOLDER uses a sequencealignment between a template and model protein to identify residues intopologically equivalent positions. For topologically nonequivalentatoms, such as variable loops and some side chains, chemicalconstraints, standard geometrical parameters, and chemical informationlike disulfide cross-links are used to compute a set of distancesbetween these atoms, which is appended to the set of distances fortopologically equivalent atoms. The x-ray crystallographic coordinatesfor M-CSF (Pandit et al., Science 258:1358-1362 (1992) served as thestructural template for modelling flt3-L, because M-CSF is the mostclosely related protein to flt3-L for which a crystal structure has beensolved. Flt3-L was modeled with the assumption that the dimer interfacewould be the same in M-CSF and flt3-L. The computer graphics programInsight was used to generate the images shown in FIG. 4.

[0204]FIG. 4 is a stereo diagram of an α-carbon ribbon trace of flt3-Lgold. One subunit is represented in gray, and the other subunit is shownin brown. The axis of dyad symmetry runs approximately horizontal in theplane of the page. The helices of the lower subunit are color coded; thetwo front helices, A and D are yellow, and the two back helices, B andC, are gold. The α-carbon of specific residues are represented as ballsand are color coded; cysteine residues are light yellow, flt3-L-residues are red, and flt3-L⁺⁺ residues are blue. Position 8 is coloredblue, although a flt3-L⁻ mutation also occurs at this site. Flt3-Lmutations listed in Table 1 whose activity is greater than wild type orreduced more than 75% of wild type are represented, except the cysteinesubstitutions R20C and R55C. Position labels represent those proteinsthat were purified. FIG. 4B is a space-filling model of flt3-L.Orientation, coloration (except helices, which are uniform gold),representation, and numbering are as in FIG. 4A. Flt3L mutations listedin Table 1 whose activity is greater than wild type or reduced more than75% of wild type are represented and labeled.

[0205] As shown in the model, the L27P mutation (SEQ ID NO:13) maps tothe predicted dimerization interface of flt3-L. Mutations at thedimerization interface may destabilize flt3-L dimers, resulting in amonomeric flt3-L. Analysis of L27P by size exclusion chromatographyindicated that it is dimeric at 0.1 mg/ml (Table 2). To test whethermonomeric flt3-L species would be observed at lower concentrations, L27Pand wild type flt3-L were diluted and analyzed by size exclusionchromatography. See FIG. 5A (wild type flt3-L) and 5B (L27P). Theconcentration of flt3-L proteins was 0.28 mg/ml (solid line) or 0.017mg/ml (dashed line). The detection wavelength was set at 220 nm. A 50-μlinjection volume was used, and the low protein concentration peakprofile was scaled to allow comparison of elution times from differentprotein concentrations.

[0206] As shown in FIG. 5A, wild type flt3-L eluted at 8.1 min at bothconcentrations, i.e., at 0.28 or 0.017 mg/ml. At 0.28 mg/ml, the elutiontime of the L27P protein was nearly identical to the wild type dimericflt3-L. However, as the concentration of the L27P protein was reduced to0.017 mg/ml, the peak at 8.1 min was reduced in size, and a second peakwas observed at 8.6 min. (FIG. 5B). The change in the elution time ofthe L27P protein when diluted from 0.28 mg/ml to 0.017 mg/ml correspondsto a shift in the observed molecular weight from 44,000 daltons to28,000 daltons. In contrast, the wild type flt3-L protein remains at44,000 daltons for both concentrations. These data indicate that theL27P mutation, which produces a mutant protein that is expressed at highlevels, alters the flt3-L dimerization interface, resulting in monomericflt3-L species at reduced protein concentrations. The fact that the L27Pmutation maps to the predicted dimerization interface and altersmonomer-dimer equilibrium of the native flt3-L polypeptide validates theM-CSF polypeptide structure as a template for modeling flt3-L structure.

[0207] The mutagenesis data and the three-dimensional model of flt3-Lcorroborate each other. The three hot spot regions (positions 8-15,81-87, and 116-124 of the mature flt3-L polypeptide, SEQ ID NO:18),widely separated in the primary structure, cluster together in a smallsurface patch of the tertiary structure. The clustering of mutations ina small surface patch is consistent with a single receptor binding siteper monomer as suggested by studies of similar receptor-ligand systems.The model also indicates that some mutations that result in decreasedflt3-L biological activity map to the proposed dimer interface. Inaddition to L27P, the mutations L26F and A64T, which also map to thedimer interface were identified. Alteration of the dimerizationinterface and reduction in biological activity may be a generalphenomenon among four helix bundle cytokines. In the protein sequencealignment proposed by Hannum et al., Nature 368:643-648 (1994), thealanine at position 64 of flt3-L correlates with the Phe at position 64in SCF. A mutation at Phe⁶⁷ in SCF alters monomer-dimer equilibriumtoward monomer and reduces the biological activity of SCF. Hsu etj al.,J. Biol. Chem. 272: 6406-6415 (1997).

[0208] As noted above, mutations that map to three hot spots scatteredthroughout the primary sequence cluster together in a patch on theflt3-L three-dimensional model (FIG. 4). The histidine at position 8 ofthe mature wild type flt3-L polypeptide maps to the center of thispatch. Two substitutions for histidine at position 8 were isolated, H8R,which is flt3L⁻, and H8Y, which is flt3L⁺⁺. This histidine is conservedin murine flt3-L and M-CSF (see FIG. 1). The activity of M-CSF isreduced when the equivalent histidine, at position 9 in the M-CSFpolypeptide, is substituted by alanine. High resolutionthree-dimensional analysis of M-CSF mutant H9A/H15A shows no significantstructural perturbations. These data suggest that His⁹ of M-CSF and His⁸of flt3-L are directly involved in the binding energetics with theirrespective receptors.

[0209] The positively charged lysine at position 84 of the mature wildtype flt3-L polypeptide is the penultimate residue of the C terminus ofhelix C. In mutants K84E and K84T, the substitution of this lysine withnonbasic residues results in a flt3-L⁺⁺ phenotype. The threoninesubstitution of K84T is conservative with the serines found in theequivalent site in M-CSF and murine flt3-L, indicating that Lys⁸⁴ actsto diminish activity in native flt3-L by destabilizing the interactionwith flt3.

[0210] Inspection of FIG. 5B reveals that K84E, Q122R, and H8Y form atriangle of residues that enhance binding of flt3-L to its receptor.Residues that map within this triangle include Asp³, Cys⁴, Ser⁵, andGln⁷. Other residues that map within the triangle include Ser⁵ and Gln⁷,which are Tyr⁵ and Ser⁷ in murine flt3-L, respectively (FIG. 1). Sincemurine flt3-L stimulates human flt3, these residues are not critical forreceptor binding. These data indicate that residues that can be changedto increase receptor affinity also include residues that are not part ofa continuous patch, i.e., they are not directly involved in receptorbinding. Consistent with this interpretation is the observation thatW118R and L-3H increase binding affinity for the flt3 receptor andintroduce an extra positive charge to the molecule. The W118R mutationdisrupts some of the helical content of the molecule (Table 2), andLeu⁻³ is a residue that is not normally found in the mammalian expressedmolecule, so it is unlikely that either residue is part of the flt3binding site. Thus, the introduction of additional basic residues intothe wild type flt3-L polypeptide, or replacement of residues in flt3-Lwith basic residues, can be used to generate flt3-L mutant polypeptideswith increased biological activity.

Example 7

[0211] Use of Flt3-L Mutants in Peripheral Stem Cell Transplantation

[0212] The flt3-L mutant polypeptides described herein have alteredflt3-L biological activity. Some flt3-L mutant polypeptides have greaterflt3-L biological activity than wild type flt3-L, as measured byinducement of cellular proliferation. These mutants would therefore beuseful in methods of cell expansion and transplantation. In particular,flt3-L mutants exhibiting increased biological activity can be used toexpand cells for autologous peripheral stem cell (PSC) or peripheralblood progenitor cell (PBPC) transplantation. Methods for performingthese procedures are described in detail in U.S. Pat. No. 5,554,512,hereby incorporated by reference.

[0213] PBPC or PSC may be mobilized or increased prior to collection ofcells from a patient. This mobilization or increase can be effected bythe intravenous administration of a pharmaceutical preparation of flt3-Lmutant protein prior to cell collection. Other growth factors such asM-CSF, GM-CSF, SCF, G-CSF, EPO, TPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL14, IL-15, GM-CSF/IL-3fusion proteins, LIF, FGF and combinations thereof, can be administeredsequentially or concurrently with the flt3-L mutant polypeptide.

[0214] Mobilized or non-mobilized PBPC and PSC are collected using knownapheresis procedures. See, e.g., Bishop et al., Blood, vol. 83, No. 2,pp. 610-616 (1994). Briefly, PBPC and PSC are collected usingconventional devices, such as a Haemonetics Model V50 apheresis device(Haemonetics, Braintree, Mass.). Four-hour collections are performedtypically no more than five times weekly until approximately 6.5×10⁸mononuclear cells (MNC)/kg patient are collected. Aliquots of collectedPBPC and PSC are assayed for granulocyte-macrophage colony-forming unit(CFU-GM) content by diluting the cells approximately 1:6 with Hank'sbalanced salt solution without calcium or magnesium (HBSS) and layeringover lymphocyte separation medium (Organon Teknika, Durham, N.C.).Following centrifugation, MNC at the interface are collected, washed andresuspended in HBSS. One milliliter aliquots containing approximately300,000 MNC, modified McCoy's 5A medium, 0.3% agar, 200 U/mL recombinanthuman GM-CSF, 200 u/mL recombinant human IL-3, and 200 u/mL recombinanthuman G-CSF are cultured at 37° C. in 5% CO₂ in fully humidified air for14 days. Optionally, a flt3-L mutant polypeptide or GM-CSF/IL-3 fusionmolecules (PIXY 321) may be added to the cultures. These cultures arestained with Wright's stain, and CFU-GM colonies are scored using adissecting microscope (Ward et al., Exp. Hematot., 16:358 (1988)).Alternatively, CFU-GM colonies can be assayed using, the CD34/CD33 flowcytometry method of Siena et al., Blood, Vol. 77, No. 2, pp 400-409(1991), or any other method known in the art.

[0215] CFU-GM containing cultures can be frozen in a controlled ratefreezer (e.g., Cryo-Med, Mt. Clemens, Mich.), then stored in the vaporphase of liquid nitrogen. Ten percent dimethylsulfoxide can be used as acryoprotectant. After all collections from the patient have been made,CFU-GM-containing cultures are thawed and pooled. The pooled cells maybe then be expanded ex vivo, prior to intravenous infusion into apatient. Ex vivo expansion of pooled cells can be performed using amutant flt3-L polypeptide as a growth factor either alone, sequentiallyor in concurrent combination with other cytokines listed above, usingmethods that are well known in the art. To facilitate engraftment of thetransplanted cells, a mutant flt3-L polypeptide may be administeredsimultaneously with, or subsequent to, the infusion, either alone,sequentially or in concurrent combination with other cytokines selectedfrom the list above.

Example 8

[0216] Generation of Dendritic Cells in vitro

[0217] Stem cells, e.g., cells having the CD34⁺ phenotype, are isolatedas described above, for example, first by generating a buffy coat ofcells. Cells from the buffy coat are then incubated with a CD34 specificmonoclonal antibody. The selected CD34⁺ cells are then cultured inMcCoy's enhanced media with 20 ng/ml each of GM-CSF, IL4, TNF-α, or 100ng/ml flt3-L or c-kit ligand. The culture is continued for approximatelytwo weeks at 37° C. in 10% CO₂ in humid air. Cells then are sorted byflow cytometry for CD1 a⁺ and HLA-DR⁺ expression.

Example 9

[0218] Use of Flt3-L Mutant Polypeptides to Promote Dendritic CellExpansion

[0219] This Example describes a method for using flt3-L mutantpolypeptides for dendritic cell expansion. Prior to cell collection, itmay be desirable to mobilize or increase the numbers of circulating PBPCand PBSC. Mobilization can improve PBPC and PBSC collection, and isachievable through the intravenous administration of flt3-ligand orsargramostim (Leukine®, Immunex Corporation, Seattle, Wash.) to apatient prior to collection of such cells. Other growth factors such asM-CSF, GM-CSF, SCF, G-CSF, EPO, TPO, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF/IL-3fusion proteins, LIF, FGF and combinations thereof, can be likewiseadministered in sequence, or in concurrent combination with flt3-L.Mobilized or non-mobilized PBPC and PBSC are collected using apheresisprocedures known in the art. See, for example, Bishop et al., Blood,vol. 83, No. 2, pp. 610-616 (1994). Briefly, PBPC and PBSC are collectedusing conventional devices, for example, a Haemonetics Model V50apheresis device (Haemonetics, Braintree, Mass.). Four-hour collectionsare performed typically no more than five times weekly untilapproximately 6.5×10⁸ mononuclear cells (MNC)/kg patient are collected.Aliquots of collected PBPC and PBSC are assayed forgranulocyte-macrophage colony-forming unit (CFU-GM) content by dilutingapproximately 1:6 with Hank's balanced salt solution without calcium ormagnesium (HBSS) and layering over lymphocyte separation medium (OrganonTeknika, Durham, N.C.) Following centrifugation, MNC at the interfaceare collected, washed and resuspended in HBSS. One milliliter aliquotscontaining approximately 300,000 MNC, modified McCoy's 5A medium, 0.3%agar, 200 U/mL recombinant human GM-CSF, 200 u/mL recombinant humanIL-3, and 200 u/mL recombinant human G-CSF or GM-CSF/IL-3 fusionmolecules (PIXY 321) may be added to the cultures. These cultures arestained with Wright's stain, and CFU-GM colonies are scored using adissecting microscope (Ward et al., Exp. Hematol., 16:358 (1988)).Alternatively, CFU-GM colonies can be assayed using the CD34/CD33 flowcytometry method of Siena et al., Blood, Vol. 77, No. 2, pp. 400-409(1991), or any other method know in the art.

[0220] CFU-GM containing cultures are frozen in a controlled ratefreezer (e.g., Cryo-Med, Mt. Clemens, Mich.), then stored in the vaporphase of liquid nitrogen. Ten percent dimethylsulfoxide can be used as acryoprotectant. After all collections from the patient have been made,CFU-GM containing cultures are thawed and pooled. The thawed cellcollection is contacted with a flt3-L mutant polypeptide, either alone,sequentially or in concurrent combination with other cytokines listedabove. Such exposure to flt3-L mutant polypeptides will drive the CFU-GMto differentiate into dendritic cells. The expanded dendritic cellpopulation is then administered to the patient, e.g., intravenously.

1 20 1 235 PRT Homo sapiens 1 Met Thr Val Leu Ala Pro Ala Trp Ser ProThr Thr Tyr Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Ser Ser Gly Leu SerGly Thr Gln Asp Cys Ser Phe 20 25 30 Gln His Ser Pro Ile Ser Ser Asp PheAla Val Lys Ile Arg Glu Leu 35 40 45 Ser Asp Tyr Leu Leu Gln Asp Tyr ProVal Thr Val Ala Ser Asn Leu 50 55 60 Gln Asp Glu Glu Leu Cys Gly Gly LeuTrp Arg Leu Val Leu Ala Gln 65 70 75 80 Arg Trp Met Glu Arg Leu Lys ThrVal Ala Gly Ser Lys Met Gln Gly 85 90 95 Leu Leu Glu Arg Val Asn Thr GluIle His Phe Val Thr Lys Cys Ala 100 105 110 Phe Gln Pro Pro Pro Ser CysLeu Arg Phe Val Gln Thr Asn Ile Ser 115 120 125 Arg Leu Leu Gln Glu ThrSer Glu Gln Leu Val Ala Leu Lys Pro Trp 130 135 140 Ile Thr Arg Gln AsnPhe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro 145 150 155 160 Asp Ser SerThr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175 Thr AlaPro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu 180 185 190 ProVal Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln 195 200 205Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro Val 210 215220 Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His 225 230 235 2 988 DNAHomo sapiens CDS (30)..(734) 2 cggccggaat tccggggccc ccggccgaa atg acagtg ctg gcg cca gcc tgg 53 Met Thr Val Leu Ala Pro Ala Trp 1 5 agc ccaaca acc tat ctc ctc ctg ctg ctg ctg ctg agc tcg gga ctc 101 Ser Pro ThrThr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu 10 15 20 agt ggg acccag gac tgc tcc ttc caa cac agc ccc atc tcc tcc gac 149 Ser Gly Thr GlnAsp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp 25 30 35 40 ttc gct gtcaaa atc cgt gag ctg tct gac tac ctg ctt caa gat tac 197 Phe Ala Val LysIle Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr 45 50 55 cca gtc acc gtggcc tcc aac ctg cag gac gag gag ctc tgc ggg ggc 245 Pro Val Thr Val AlaSer Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly 60 65 70 ctc tgg cgg ctg gtcctg gca cag cgc tgg atg gag cgg ctc aag act 293 Leu Trp Arg Leu Val LeuAla Gln Arg Trp Met Glu Arg Leu Lys Thr 75 80 85 gtc gct ggg tcc aag atgcaa ggc ttg ctg gag cgc gtg aac acg gag 341 Val Ala Gly Ser Lys Met GlnGly Leu Leu Glu Arg Val Asn Thr Glu 90 95 100 ata cac ttt gtc acc aaatgt gcc ttt cag ccc ccc ccc agc tgt ctt 389 Ile His Phe Val Thr Lys CysAla Phe Gln Pro Pro Pro Ser Cys Leu 105 110 115 120 cgc ttc gtc cag accaac atc tcc cgc ctc ctg cag gag acc tcc gag 437 Arg Phe Val Gln Thr AsnIle Ser Arg Leu Leu Gln Glu Thr Ser Glu 125 130 135 cag ctg gtg gcg ctgaag ccc tgg atc act cgc cag aac ttc tcc cgg 485 Gln Leu Val Ala Leu LysPro Trp Ile Thr Arg Gln Asn Phe Ser Arg 140 145 150 tgc ctg gag ctg cagtgt cag ccc gac tcc tca acc ctg cca ccc cca 533 Cys Leu Glu Leu Gln CysGln Pro Asp Ser Ser Thr Leu Pro Pro Pro 155 160 165 tgg agt ccc cgg cccctg gag gcc aca gcc ccg aca gcc ccg cag ccc 581 Trp Ser Pro Arg Pro LeuGlu Ala Thr Ala Pro Thr Ala Pro Gln Pro 170 175 180 cct ctg ctc ctc ctactg ctg ctg ccc gtg ggc ctc ctg ctg ctg gcc 629 Pro Leu Leu Leu Leu LeuLeu Leu Pro Val Gly Leu Leu Leu Leu Ala 185 190 195 200 gct gcc tgg tgcctg cac tgg cag agg acg cgg cgg agg aca ccc cgc 677 Ala Ala Trp Cys LeuHis Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg 205 210 215 cct ggg gag caggtg ccc ccc gtc ccc agt ccc cag gac ctg ctg ctt 725 Pro Gly Glu Gln ValPro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu 220 225 230 gtg gag cactgacctggcc aaggcctcat cctgcggagc cttaaacaac 774 Val Glu His 235gcagtgagac agacatctat catcccattt tacaggggag gatactgagg cacacagagg 834ggagtcacca gccagaggat gtatagcctg gacacagagg aagttggcta gaggccggtc 894ccttccttgg gcccctctca ttccctcccc agaatggagg caacgccaga atccagcacc 954ggccccattt acccaactct gaacaaagcc cccg 988 3 40 DNA Artificial SequenceDescription of Artificial Sequence primer 3 attaggtacc tttggataaaagactcagtg ggaccaggac 40 4 37 DNA Artificial Sequence Description ofArtificial Sequence primer 4 atatggatcc ctacggggct gtggcctcca ggggccg 375 77 DNA Artificial Sequence Description of Artificial Sequence primer 5cctcctgcag gagacctccg agcagctggt ggcgctgaag ccctggatca ctcgccagaa 60cttcgcccgg tgcctgg 77 6 46 DNA Artificial Sequence Description ofArtificial Sequence constructed oligonucleotide 6 tggataaaag acacagtgggacccaggact gctccttcca atacag 46 7 46 DNA Artificial Sequence Descriptionof Artificial Sequence constructed oligonucleotide 7 tggataaaagacacagtggg acccaggact gctccttcca atacag 46 8 209 PRT Homo sapiens 8 ThrGln Asp Cys Ser Phe Gln Arg Ser Pro Ile Ser Ser Asp Phe Ala 1 5 10 15Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 65 70 7580 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 9095 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro TrpSer 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln ProPro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu LeuLeu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln Arg Thr Arg Arg ArgThr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro Val Pro Ser Pro GlnAsp Leu Leu Leu Val Glu 195 200 205 His 9 209 PRT Homo sapiens 9 Thr GlnAsp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala 1 5 10 15 ValLys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30 ThrVal Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45 ArgLeu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Thr 50 55 60 GlySer Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 65 70 75 80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe 85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu 100 105110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu 115120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro ProLeu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu LeuAla Ala Ala 165 170 175 Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg ThrPro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro Val Pro Ser Pro Gln AspLeu Leu Leu Val Glu 195 200 205 His 10 212 PRT Homo sapiens 10 His SerGly Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser 1 5 10 15 AspPhe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp 20 25 30 TyrPro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly 35 40 45 GlyLeu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys 50 55 60 ThrVal Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr 65 70 75 80Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys 85 90 95Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser 100 105110 Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser 115120 125 Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro130 135 140 Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala ProGln 145 150 155 160 Pro Pro Leu Leu Leu Leu Leu Leu Leu Pro Val Gly LeuLeu Leu Leu 165 170 175 Ala Ala Ala Trp Cys Leu His Trp Gln Arg Thr ArgArg Arg Thr Pro 180 185 190 Arg Pro Gly Glu Gln Val Pro Pro Val Pro SerPro Gln Asp Leu Leu 195 200 205 Leu Val Glu His 210 11 209 PRT Homosapiens 11 Thr Gln Asp Cys Ser Phe Gln Tyr Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 12 209 PRT Homosapiens 12 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Phe Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 13 209 PRT Homosapiens 13 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Pro Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 14 209 PRT Homosapiens 14 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Glu Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 15 209 PRT Homosapiens 15 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Thr Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 16 209 PRT Homosapiens 16 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Arg Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 17 209 PRT Homosapiens 17 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Arg Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 18 209 PRT Homosapiens 18 Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp PheAla 1 5 10 15 Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp TyrPro Val 20 25 30 Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly GlyLeu Trp 35 40 45 Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys ThrVal Ala 50 55 60 Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr GluIle His 65 70 75 80 Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser CysLeu Arg Phe 85 90 95 Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr SerGlu Gln Leu 100 105 110 Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn PheSer Arg Cys Leu 115 120 125 Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr LeuPro Pro Pro Trp Ser 130 135 140 Pro Arg Pro Leu Glu Ala Thr Ala Pro ThrAla Pro Gln Pro Pro Leu 145 150 155 160 Leu Leu Leu Leu Leu Leu Pro ValGly Leu Leu Leu Leu Ala Ala Ala 165 170 175 Trp Cys Leu His Trp Gln ArgThr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190 Glu Gln Val Pro Pro ValPro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200 205 His 19 137 PRTMurine 19 Thr Pro Asp Cys Tyr Phe Ser His Ser Pro Ile Ser Ser Asn PheLys 1 5 10 15 Val Lys Phe Arg Glu Leu Thr Asp His Leu Leu Lys Asp TyrPro Val 20 25 30 Thr Val Ala Val Asn Leu Gln Asp Glu Glu Lys His Cys LysAla Leu 35 40 45 Trp Ser Leu Phe Leu Ala Gln Arg Trp Ile Glu Gln Leu LysThr Val 50 55 60 Ala Gly Ser Lys Met Gln Thr Leu Leu Glu Asp Val Asn ThrGlu Ile 65 70 75 80 His Phe Val Thr Ser Cys Thr Phe Gln Pro Leu Pro GluCys Leu Arg 85 90 95 Phe Val Gln Thr Asn Ile Ser His Leu Leu Lys Asp ThrCys Thr Gln 100 105 110 Leu Leu Ala Leu Lys Pro Cys Ile Gly Lys Ala CysGln Asn Phe Ser 115 120 125 Arg Cys Leu Glu Val Gln Cys Gln Pro 130 13520 149 PRT Homo sapiens 20 Glu Glu Val Ser Glu Tyr Cys Ser His Met IleGly Ser Gly His Leu 1 5 10 15 Gln Ser Leu Gln Arg Leu Ile Asp Ser GlnMet Glu Thr Ser Cys Gln 20 25 30 Ile Thr Phe Glu Phe Val Asp Gln Glu GlnLeu Lys Asp Pro Val Cys 35 40 45 Tyr Leu Lys Lys Ala Phe Leu Leu Val GlnAsp Ile Met Glu Asp Thr 50 55 60 Met Arg Phe Arg Asp Asn Thr Pro Asn AlaIle Ala Ile Val Gln Leu 65 70 75 80 Gln Glu Leu Ser Leu Arg Leu Lys SerCys Phe Thr Lys Asp Tyr Glu 85 90 95 Glu His Asp Lys Ala Cys Val Arg ThrPhe Tyr Glu Thr Pro Leu Gln 100 105 110 Leu Leu Glu Lys Val Lys Asn ValPhe Asn Glu Thr Lys Asn Leu Leu 115 120 125 Asp Lys Asp Trp Asn Ile PheSer Lys Asn Cys Asn Asn Ser Phe Ala 130 135 140 Glu Cys Ser Ser Gln 145

What is claimed is:
 1. A soluble mutant flt3 ligand (flt3-L)polypeptide, wherein said polypeptide exhibits increased or decreasedbiological activity relative to the full length human wild type flt3-Lpolypeptide (SEQ ID NO:1) or mature flt3-L polypeptide (SEQ ID NO:18).2. The polypeptide of claim 1, wherein said polypeptide comprises one ormore amino substitutions in any one of the regions defined by the aminoacid positions 8-15, 81-87, or 116-124 of the mature human wild typeflt3-L polypeptide (SEQ ID NO:18).
 3. The polypeptide of claim 1,wherein said polypeptide comprises one or more substitutions at position8, 84, 118 or 122 of the mature wild type flt3-L polypeptide (SEQ IDNO:18).
 4. The polypeptide of claim 1, wherein said polypeptidecomprises one or more substitutions selected from the group consistingof L-3H (SEQ ID NO:10), H8Y (SEQ ID NO:11), W118R (SEQ ID NO:16), K84E(SEQ ID NO:14), K84T (SEQ ID NO:15) and Q122R (SEQ ID NO:17).
 5. Thepolypeptide of claim 4, wherein said polypeptide comprises the L-3H (SEQID NO:10), H8Y (SEQ ID NO:11), K84E (SEQ ID NO:14) and Q122R (SEQ IDNO:17) substitutions.
 6. The polypeptide of claim 1, wherein saidpolypeptide is a fusion protein with a second polypeptide, wherein saidsecond polypeptide is selected from the group consisting oferythropoietin (EPO), thrombopoietin (TPO), granulocyte-macrophageColony Stimulating Factor (GM-CSF), granulocyte Colony StimulatingFactor (G-CSF), an interleukin, immunoglobulin, and fragments thereof.7. The polypeptide of claim 2, wherein said polypeptide is a fusionprotein with a second polypeptide, wherein said second polypeptide isselected from the group consisting of erythropoietin (EPO),thrombopoietin (TPO), granulocyte-macrophage Colony Stimulating Factor(GM-CSF), granulocyte Colony Stimulating Factor (G-CSF), an interleukin,immunoglobulin, and fragments thereof.
 8. The polypeptide of claim 3,wherein said polypeptide is a fusion protein with a second polypeptide,wherein said second polypeptide is selected from the group consisting oferythropoietin (EPO), thrombopoietin (TPO), granulocyte-macrophageColony Stimulating Factor (GM-CSF), granulocyte Colony StimulatingFactor (G-CSF), an interleukin, immunoglobulin, and fragments thereof.9. The polypeptide of claim 4, wherein said second polypeptide isselected from the group consisting of erythropoietin (EPO),thrombopoietin (TPO), granulocyte-macrophage Colony Stimulating Factor(GM-CSF), granulocyte Colony Stimulating Factor (G-CSF), an interleukin,immunoglobulin, and fragments thereof.
 10. The polypeptide of claim 5,wherein said second polypeptide is selected from the group consisting oferythropoietin (EPO), thrombopoietin (TPO), granulocyte-macrophageColony Stimulating Factor (GM-CSF), granulocyte Colony StimulatingFactor (G-CSF), an interleukin, immunoglobulin, and fragments thereof.11. The polypeptide of claim 1, wherein said polypeptide comprises amutation at the dimerization interface of a flt3-L dimer.
 12. Thepolypeptide of claim 11, wherein said mutation is at position 26, 27 or64 of the mature wild type flt3-L polypeptide (SEQ ID NO:18).
 13. Thepolypeptide of claim 11, wherein said polypeptide comprises a mutationselected from the group consisting of L26F (SEQ ID NO:12), L27P (SEQ IDNO:13) or A64T (SEQ ID NO:9).
 14. The polypeptide of claim 2, furthercomprising a mutation at the dimerization interface of a flt3-Lpolypeptide.
 15. The polypeptide of claim 1, wherein said polypeptidehas an altered charge distribution from that of the wild type humanflt3-L full length polypeptide (SEQ ID NO:1) or mature human flt3-Lpolypeptide (SEQ ID NO:18).
 16. The polypeptide of claim 15, wherein atleast one amino acid of the wild type flt3-L polypeptide has beensubstituted by a basic residue.
 17. The polypeptide of claim 16, whereinsaid substitution occurs in the region corresponding to positions118-124 of the mature wild type flt3-L polypeptide (SEQ ID NO:18). 18.The polypeptide of claim 17, wherein said substitution is at position118 or position 122 of the mature wild type flt3-L polypeptide (SEQ IDNO:18).
 19. The polypeptide of claim 15, wherein at least one basicresidue has been added to the full length wild type flt3-L polypeptide(SEQ ID NO:1) or the mature wild type human flt3-L polypeptide (SEQ IDNO:18).
 20. The polypeptide of claim 15, wherein a basic amino acid ofwild type flt3-L has been replaced with another amino acid.
 21. Thepolypeptide of claim 19, wherein said basic amino acid is the Lys atposition 84 of mature human wild type flt3-L (SEQ ID NO:18).
 22. Thepolypeptide of claim 1, wherein said mutant flt3-L polypeptide comprisesamino acids 28-160, 28-182 or 28-185 of the full length human wild typeflt3-L polypeptide (SEQ ID NO:1).
 23. An isolated nucleic acid encodinga polypeptide of claim
 1. 24. An isolated nucleic acid encoding apolypeptide of claim
 2. 25. An isolated nucleic acid encoding apolypeptide of claim
 3. 26. An isolated nucleic acid encoding apolypeptide of claim
 4. 27. An isolated nucleic acid encoding apolypeptide of claim
 5. 28. An isolated nucleic acid encoding apolypeptide of claim
 6. 29. An isolated nucleic acid encoding apolypeptide of claim
 7. 30. An isolated nucleic acid encoding apolypeptide of claim
 8. 31. An isolated nucleic acid encoding apolypeptide of claim
 9. 32. An isolated nucleic acid encoding apolypeptide of claim
 10. 33. A method of inducing cellular expansion,comprising the steps of: isolating a population of cells to be expanded;and exposing said cells to a mutant flt3-L polypeptide, to produce anexpanded cell population.
 34. The method of claim 33, wherein theexpanded cell population is introduced into a patient.
 35. The method ofclaim 33, wherein the population of cells to be expanded compriseshematopoietic cells.
 36. The method of claim 33, wherein the populationof cells is also exposed to a growth factor in addition to said flt3-Lmutant polypeptide.
 37. The method of claim 33, wherein said growthfactor is selected from the group consisting of interleukins, colonystimulating factors, and protein kinases.
 38. A method of expanding apopulation of cells in vivo, comprising the step of administering to asubject a pharmaceutical composition of mutant flt3-L polypeptide ornucleic acid encoding such polypeptide sufficient to induce theexpansion of a target cell population.
 39. The method of claim 38,wherein the target cell population is isolated from the group consistingof hematopoietic cells, NK cells or dendritic cells.
 40. The method ofclaim 38, wherein the pharmaceutical composition further comprises agrowth factor in addition to said flt3-L mutant polypeptides.
 41. Themethod of claim 40, wherein said growth factor is selected from thegroup consisting of interleukins, colony stimulating factors and proteinkinases.
 42. A method of modulating an immune response in a subject,said method comprising administering to said subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a flt3-Lmutant polypeptide or nucleic acid encoding such polypeptide.
 43. Amethod of treating an immune disorder in a subject, said methodcomprising administering to said subject a therapeutically effectiveamount of a pharmaceutical composition comprising a flt3-L mutantpolypeptide or nucleic acid encoding such polypeptide.
 44. The method ofclaim 43, wherein said disorder is selected from the group consisting ofallergy, immunosuppression, and autoimmunity.
 45. A method of treating apathological condition, said method comprising the step ofadministration of a pharmaceutical composition of flt3-L mutantpolypeptide or nucleic acid, wherein said condition is selected from thegroup consisting of myelodysplasia, aplastic anemia, HumanImmunodeficiency Virus infection, breast cancer, lymphoma, small celllung cancer, multiple myeloma, neuroblastoma, acute leukemia, testicularcancer and ovarian cancer.
 46. A method of inducing cellulardifferentiation, said method comprising the steps of: isolating a targetpopulation of cells; and administering an amount of flt3-L mutantpolypeptide sufficient to induce the production of differentiated cells.47. The method of claim 46, wherein said target population of cellscomprises hematopoietic cells.
 48. The method of claim 47, wherein thedifferentiated cells are selected from the group consisting of NaturalKiller (NK) cells, facilitating cells, or dendritic cells.
 49. A methodof treating a patient, comprising administering to said patient thedifferentiated cells produced by the method of claim
 46. 50. The methodof claim 49, further comprising the step of administering a growthfactor to the patient.
 51. The method of claim 49, wherein said growthfactor is selected from the group consisting of interleukins, colonystimulating factors and protein kinases.
 52. A method of augmenting animmune response in a patient, comprising the step of administering anamount of a flt3-L mutant polypeptide to the patient sufficient togenerate an increase in the number of the patient's dendritic cells. 53.The method of claim 52, wherein the patient has an infectious disease.54. The method of claim 53, wherein the infectious disease is HIV. 55.The method of claim 52, wherein the patient has a cancerous orneoplastic disease.
 56. A method of enhancing a mammal's immune responseto a vaccine antigen, comprising the steps of administering to saidmammal an immunogenic amount of the vaccine antigen and animmunogenicity-augmenting amount of a flt3-L mutant polypeptide inconcurrent or sequential combination with said vaccine antigen.
 57. Amethod for identifying residues involved in receptor binding in areceptor-ligand system, said method comprising the steps of: subjectinga nucleic acid population encoding said ligand to mutagenesis, to form amutagenized ligand population; transforming cells with said mutagenizedligand population, to form transformed colonies; transferring saidtransformed colonies to a first membrane; overlaying said first membranewith a second membrane, said second membrane being coated with capturemeans for capturing said ligand and mutants thereof; reacting saidsecond membrane with a receptor for said ligand; and subsequentlyreacting said second membrane with means for detecting receptor bindingto said ligand or mutants thereof.
 58. The method of claim 57, whereinsaid cells are selected from the group consisting of yeast cells andbacterial cells.
 59. A method of screening to identify mutantpolypeptides with altered expression characteristics, said methodcomprising the steps of: subjecting a nucleic acid population encodingsaid ligand to mutagenesis, to form a mutagenized ligand population;transforming cells with said mutagenized ligand population, to formtransformed colonies; transferring said transformed colonies to a firstmembrane; overlaying said first membrane with a second membrane, saidsecond membrane being coated with capture means for capturing saidligand and mutants thereof; reacting said second membrane with areceptor for said ligand; and subsequently reacting said second membranewith means for detecting receptor binding to said ligand or mutantsthereof.
 60. The method of claim 59, wherein said cells are selectedfrom the group consisting of yeast cells and bacteria cells.
 61. Amutant Stem Cell Factor (SCF) or Macrophage Colony Stimulating Factor(M-CSF) polypeptide, wherein said polypeptide has an amino acidsubstitution at a position of said polypeptide corresponding to any oneof the regions defined by the amino acid positions 8-15, 81-97 or116-124 of the mature human wild type flt3-L polypeptide (SEQ ID NO:18)and exhibits increased binding to a flt3 polypeptide compared to wildtype SCF or M-CSF.
 62. The polypeptide of claim 61, wherein saidpolypeptide has an amino acid substitution at the position correspondingto position 8, 84, 118 or 122 of the mature human wild type flt3-Lpolypeptide (SEQ ID NO:18).
 63. The polypeptide of claim 61, whereinsaid polypeptide has an amino acid substitution at the positioncorresponding to position 8 of the mature human wild type flt3-Lpolypeptide (SEQ ID NO:18).
 64. The polypeptide of claim 63, whereinsaid mutant polypeptide is a SCF mutant polypeptide.
 65. The polypeptideof claim 64, wherein said polypeptide does not bind c-kit.
 66. Thepolypeptide of claim 64, wherein said polypeptide does not bind to mastcells.
 67. The mutant polypeptide of claim 61, wherein said mutantpolypeptide is M-CSF and has an amino acid substitution at position 9 ofthe wild type M-CSF polypeptide.
 68. A small molecule comprising any oneof the regions defined by the amino acid positions 8-15, 81-87 or116-124 of the mature human wild type flt3-L polypeptide (SEQ ID NO:18),or functional groups corresponding to the side chains of the amino.